Download ABI Prism® 7900HT Sequence Detection System
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ABI PRISM 7900HT ® Sequence Detection System User Guide Basic Operation and Maintenance ABI PRISM 7900HT ® Sequence Detection System User Guide © Copyright 2001, Applied Biosystems. All Rights Reserved. For Research Use Only. Not for use in diagnostic procedures. ABI PRISM and its design, AmpliCover and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. ABI, Applied Biosystems, FAM, JOE, NED, ROX, TAMRA, TET, VIC, and Primer Express are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. AmpErase, AmpliTaq, AmpliTaq Gold, GeneAmp, SYBR, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. Windows and Microsoft are registered trademarks of Microsoft, Inc. Twister Universal Plate Handler is a trademark of Zymark Corporation. Twister Users Guide (part number 66503 from Zymark Corporation) All other trademarks are the sole property of their respective owners. Applera Corporation is committed to providing the world’s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Authorized Thermal Cycler This ABI PRISM® 7900 HT Sequence Detection System Base Unit, Serial No___________, in combination with its immediately attached sample block modules, comprise an Authorized Thermal Cycler. The purchase price of this Base Unit includes the up_front fee component of a license under United States Patent Nos. 4,683,195, 4,683,202 and 4,965,188, owned by Roche Molecular Systems, Inc., and under corresponding claims in patents outside the United States, owned by F. Hoffmann_La Roche Ltd, covering the Polymerase Chain Reaction ("PCR") process to practice the PCR process for internal research and development using this instrument. The running royalty component of that license may be purchased from Applied Biosystems or obtained by purchasing Authorized Reagents. This instrument is also an Authorized Thermal Cycler for use with applications licenses available from Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Purchase of this product does not itself convey to the purchaser a complete license or right to perform the PCR process. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404. DISCLAIMER OF LICENSE: No rights for any application, including any in vitro diagnostic application, are conveyed expressly, by implication or by estoppel under any patent or patent applications claiming homogeneous or real_time detection methods, including patents covering such methods used in conjunction with the PCR process or other amplification processes. The 5' nuclease detection assay and certain other homogeneous or real_time amplification and detection methods are covered by United States Patent Nos. 5,210,015, 5,487,972, 5,804,375 and 5,994,056, owned by Roche Molecular Systems, Inc.; by corresponding patents and patent applications outside the United States, owned by F. Hoffmann_La Roche Ltd; and by United States Patent Nos. 5,538,848 and 6,030,787, and corresponding patents and patent applications outside the United States, owned by Applera Corporation. Purchase of this instrument conveys no license or right under the foregoing patents. Use of these and other patented processes in conjunction with the PCR process requires a license. For information on obtaining licenses, contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, or The Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California, 94501 JpegEncoder Licensing Statement The JpegEncoder and its associated classes are Copyright (c) 1998, James R. Weeks and BioElectroMech. This software is based in part on the work of the Independent JPEG Group. Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met: 1. Redistributions of source code must retain the above copyright notice, this list of conditions, all files included with the source code, and the following disclaimer. 2. Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution. THIS SOFTWARE IS PROVIDED BY THE AUTHOR AND CONTRIBUTORS ``AS IS'' AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE AUTHOR OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE. Contents 1 Safety Attention Words and Warning Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Chemical Hazards, Waste Profiles, and Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Obtaining Material Safety Data Sheets (MSDS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5 Safe Instrument Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6 2 Product Overview System Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Section: Getting to Know the Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4 Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7 Bar Code Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8 Zymark Twister Microplate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9 Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10 Instrument Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11 Section: Getting to Know the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13 Sequence Detection System Software Components and Features. . . . . . . . . . . . . . . . . 2-14 Managing Sequence Detection System Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15 Working with SDS Software Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16 3 Getting Started Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2 About This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3 Turning on the ABI PRISM 7900HT Sequence Detection System. . . . . . . . . . . . . . . . . . 3-5 Using the SDS Software Workspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Basic Software Skills Tutorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11 Using SDS Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21 4 Run Setup and Basic Operation Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2 Setup Checklists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3 iii Section: Plate Document Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-5 Step 1 – Creating a Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6 Step 2 – Applying Detectors and Markers to the Plate Document . . . . . . . . . . . . . . . . . 4-7 Step 3 – Configuring the Plate Document with Detector Tasks . . . . . . . . . . . . . . . . . . 4-11 Step 4 – Programming the Plate Document Method . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13 Step 5 – Saving the Plate Document as a Template . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-17 Step 6 – Creating a Plate Document from the Template. . . . . . . . . . . . . . . . . . . . . . . . 4-18 Step 7 – Applying Sample and Plate Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-19 Step 8 – Running the Plate on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . 4-20 Section: Running an Individual Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-21 Saving the Plate Document. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-22 Preparing and Running a Single Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23 Operating the 7900HT Instrument Using the SDS Software . . . . . . . . . . . . . . . . . . . . 4-25 After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-27 Section: Running Multiple Plates Using the Automation Controller . . . . . . . . . . .4-29 Adding Plate Documents to the Plate Queue for Automated Operation . . . . . . . . . . . 4-30 Adding a Plate Document to the Plate Queue from the SDS Software . . . . . . . . . . . . 4-31 Creating Plate Documents Using the Template Batch Utility . . . . . . . . . . . . . . . . . . . 4-32 Running Plates Using the Automation Controller Software. . . . . . . . . . . . . . . . . . . . . 4-34 Loading Plates onto the Automation Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-36 Operating the 7900HT Instrument Using the Automation Controller Software. . . . . . 4-38 After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-38 5 End-Point Analysis End-Point Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2 Section: Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-3 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4 Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7 Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-8 Analyzing a Completed Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9 Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . . . . . 5-10 After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15 6 Real-Time Analysis Real-Time Runs on the 7900HT Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2 Section: Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-3 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4 Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6 Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7 Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8 Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13 After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15 iv Section: Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-17 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-18 Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19 Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20 Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21 Determining Tm Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22 After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-24 7 System Maintenance Recommended Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2 Section: Maintaining the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3 Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4 Changing the 7900HT Plate Adapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-9 Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-11 Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-13 Performing a Pure Dye Run. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-17 Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-21 Verifying Instrument Performance Using a TaqMan RNase P Plate . . . . . . . . . . . . . . . 7-24 Section: Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27 Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . . . . . 7-28 Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-32 Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-40 Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-43 Section: Maintaining the Computer and SDS Software . . . . . . . . . . . . . . . . . . . . . 7-45 General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-46 Maintaining the SDS software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-48 8 Troubleshooting Troubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2 Low Precision or Irreproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-4 Background Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-8 Pure Dye Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-10 Real-Time Runs (Quantitative PCR and Dissociation Curves) . . . . . . . . . . . . . . . . . . . 8-11 End-Point Runs (Allelic Discrimination) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-13 Software and 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14 Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader . . . . . . . . . 8-17 9 User Bulletins v A Theory of Operation Fluorescent-Based Chemistries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2 Fluorescence Detection and Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-4 Mathematical Transformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5 Real-Time Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7 B Importing and Exporting Plate Document Data Importing Plate Document Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2 Setup Table File Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-4 Exporting Graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-8 Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-9 C Designing TaqMan Assays Assay Development Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2 Design Tips for Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-5 Design Tips for Quantitative PCR Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6 D Kits, Reagents and Consumables E References F Contacting Technical Support G Limited Warranty Statement Index vi Safety 1 1 In This Chapter This chapter discusses the following topics: Topic Attention Words and Warning Labels See Page 1-2 Chemical Hazards, Waste Profiles, and Disposal 1-3 Obtaining Material Safety Data Sheets (MSDS) 1-5 Safe Instrument Use 1-6 Safety 1-1 Attention Words and Warning Labels Documentation User Five user attention words appear in the text of all Applied Biosystems user Attention Words documentation. Each word implies a particular level of observation or action as described below. Note Calls attention to useful information. IMPORTANT Indicates information that is necessary for proper instrument operation. ! CAUTION Indicates a potentially hazardous situation which, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. ! WARNING Indicates a potentially hazardous situation which, if not avoided, could result in death or serious injury. ! DANGER Indicates an imminently hazardous situation which, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. Instrument Safety labels are located on the instrument. Each safety label has three parts: Safety Labels ♦ A signal word panel, which implies a particular level of observation or action (e.g., CAUTION or WARNING). If a safety label encompasses multiple hazards, the signal word corresponding to the greatest hazard is used ♦ A message panel, which explains the hazard and any user action required ♦ A safety alert symbol, which indicates a potential personal safety hazard. See the ABI PRISM 7900HT Sequence Detection System Site Preparation and Safety Guide (P/N 4317595) for an explanation of all the safety alert symbols provided in several languages. Laser Exposure The components of the ABI PRISM® 7900HT Sequence Detection System contain several lasers including an Argon laser within the 7900HT instrument and a low power laser within each bar code reader (fixed-position and hand-held). ! WARNING LASER HAZARD. Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots. Never look directly into the laser beam. Remove jewelry and other items that can reflect the beam into your eyes. Protect others from exposure to the beam. Site Preparation and A site preparation and safety guide is a separate document sent to all customers who Safety Guide have purchased an Applied Biosystems instrument. Refer to the guide written for your instrument for information on site preparation, instrument safety, chemical safety, and waste profiles. 1-2 Safety Chemical Hazards, Waste Profiles, and Disposal Chemical Hazard ! WARNING CHEMICAL HAZARD. Some of the chemicals used with Applied Biosystems Warning instruments and protocols are potentially hazardous and can cause injury, illness, or death. ♦ Read and understand the material safety data sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. ♦ Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. ♦ Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (e.g., fume hood). For additional safety guidelines, consult the MSDS. ♦ Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer’s cleanup procedures as recommended on the MSDS. ♦ Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. Chemical Waste ! WARNING CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems Hazard Warning instruments are potentially hazardous and can cause injury, illness, or death. ♦ Read and understand the material safety data sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. ♦ Handle chemical wastes in a fume hood. ♦ Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. ♦ Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (e.g., fume hood). For additional safety guidelines, consult the MSDS. ♦ After emptying the waste container, seal it with the cap provided. ♦ Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. Safety 1-3 About Waste Profiles A waste profile was provided with this instrument and is contained in the ABI PRISM 7900HT Sequence Detection System Site Preparation and Safety Guide. Waste profiles list the percentage compositions of the reagents within the waste stream at installation and the waste stream during a typical user application, although this application may not be used in your laboratory. These profiles assist users in planning for instrument waste handling and disposal. Read the waste profiles and all applicable MSDSs before handling or disposing of waste. IMPORTANT Waste profiles are not a substitute for MSDS information. About Waste As the generator of potentially hazardous waste, it is your responsibility to perform the Disposal actions listed below. ♦ Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. ♦ Ensure the health and safety of all personnel in your laboratory. ♦ Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, or national regulations. Note Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. 1-4 Safety Obtaining Material Safety Data Sheets (MSDS) About MSDSs Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer. When hazards exist, warnings are prominently displayed on the labels of all chemicals. Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update. MSDSs provide you with the safety information you need to store, handle, transport and dispose of the chemicals safely. We strongly recommend that you replace the appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical. ! WARNING CHEMICAL HAZARD. Be sure to familiarize yourself with the MSDSs before using reagents or solvents. Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below. To order documents by automated telephone service: Step Action 1 From the U.S. or Canada, dial 1.800.487.6809, or from outside the U.S. and Canada, dial 1.858.712.0317. 2 Follow the voice instructions to order documents (for delivery by fax). Note There is a limit of five documents per fax request. To order documents by telephone: In the U.S. Dial 1.800.345.5224, and press 1. ♦ To order in English, dial 1.800.668.6913 and press 1, then 2, then 1. In Canada ♦ To order in French, dial 1.800.668.6913 and press 2, then 2, then 1. From any other country See the specific region under “To Contact Technical Support by Telephone or Fax (Outside North America).” To view, download, or order documents through the Applied Biosystems web site: Step Action 1 Go to http://www.appliedbiosystems.com 2 Click SERVICES & SUPPORT at the top of the page, click Documents on Demand, then click MSDS. 3 Click MSDS Index, search through the list for the chemical of interest to you, then click on the MSDS document number for that chemical to open a PDF version of the MSDS. For chemicals not manufactured or distributed by Applied Biosystems, call the chemical manufacturer. Safety 1-5 Safe Instrument Use Before Operating the Ensure that everyone involved with the operation of the instrument has: Instrument ♦ Received instruction in general safety practices for laboratories ♦ Received instruction in specific safety practices for the instrument ♦ Read and understood all related MSDSs ! CAUTION Avoid using this instrument in a manner not specified by Applied Biosystems. Although the instrument has been designed to protect the user, this protection can be impaired if the instrument is used improperly. Safe and Efficient Operating the computer correctly prevents stress-producing effects such as fatigue, Computer Use pain, and strain. To minimize these effects on your back, legs, eyes, and upper extremities (neck, shoulder, arms, wrists, hands and fingers), design your workstation to promote neutral or relaxed working positions. This includes working in an environment where heating, air conditioning, ventilation, and lighting are set correctly. See the guidelines below. ! CAUTION MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD. These hazards are caused by the following potential risk factors which include, but are not limited to, repetitive motion, awkward posture, forceful exertion, holding static unhealthy positions, contact pressure, and other workstation environmental factors. ♦ ♦ 1-6 Safety Use a seating position that provides the optimum combination of comfort, accessibility to the keyboard, and freedom from fatigue-causing stresses and pressures. – The bulk of the person’s weight should be supported by the buttocks, not the thighs. – Feet should be flat on the floor, and the weight of the legs should be supported by the floor, not the thighs. – Lumbar support should be provided to maintain the proper concave curve of the spine. Place the keyboard on a surface that provides: – The proper height to position the forearms horizontally and upper arms vertically. – Support for the forearms and hands to avoid muscle fatigue in the upper arms. ♦ Position the viewing screen to the height that allows normal body and head posture. This height depends upon the physical proportions of the user. ♦ Adjust vision factors to optimize comfort and efficiency by: – Adjusting screen variables, such as brightness, contrast, and color, to suit personal preferences and ambient lighting. – Positioning the screen to minimize reflections from ambient light sources. – Positioning the screen at a distance that takes into account user variables such as nearsightedness, farsightedness, astigmatism, and the effects of corrective lenses. ♦ When considering the user’s distance from the screen, the following are useful guidelines: – The distance from the user’s eyes to the viewing screen should be approximately the same as the distance from the user’s eyes to the keyboard. – For most people, the reading distance that is the most comfortable is approximately 20 inches. – The workstation surface should have a minimum depth of 36 inches to accommodate distance adjustment. – Adjust the screen angle to minimize reflection and glare, and avoid highly reflective surfaces for the workstation. ♦ Use a well-designed copy holder, adjustable horizontally and vertically, that allows referenced hard-copy material to be placed at the same viewing distance as the screen and keyboard. ♦ Keep wires and cables out of the way of users and passersby. ♦ Choose a workstation that has a surface large enough for other tasks and that provides sufficient legroom for adequate movement. Safety 1-7 Product Overview 2 2 In This Chapter This chapter discusses the following topics: Topic System Overview See Page 2-2 Section: Getting to Know the Hardware 2-3 7900HT Instrument 2-4 Computer 2-7 Bar Code Readers 2-8 Zymark Twister Microplate Handler 2-9 Compatible Consumables 2-10 Instrument Connections 2-11 Section: Getting to Know the Software 2-13 Sequence Detection System Software Components and Features 2-14 Managing Sequence Detection System Data 2-15 Working with SDS Software Files 2-16 Product Overview 2-1 System Overview About the 7900HT The ABI PRISM® 7900HT Sequence Detection System is a second-generation Instrument sequence detection system instrument designed for automated, high-throughput detection of fluorescent PCR-related chemistries. The instrument is capable of real-time, end-point, and dissociation curve analysis of assays arrayed on multiple formats. The 7900HT instrument is optimized for use with Applied Biosystems chemistries including those related to nucleic acid quantification and detection. Supported Runs and The ABI PRISM 7900HT Sequence Detection System is designed to support several Chemistries PCR-related chemistries available from Applied Biosystems and affiliated companies. The SDS software features two types of runs to support the variety of applications. Run/Analysis Description Supported Chemistries End Point (Plate Read) In an end-point run, the 7900HT instrument collects a single reading after completing a PCR run. After analysis by the SDS software, the resulting multicomponent data is used to assess the presence of target sequences in the unknown samples. ♦ Allelic Discrimination Real Time In an real-time run, the 7900HT instrument collects data during each cycle of a pre-programmed PCR run. After analysis by the SDS software, the resulting threshold cycle values (CT) are used to establish quantitative relationships between the initial template concentrations of the unknowns and those of the controls. ♦ Absolute Quantification ♦ Dissociation Curve Analysisa a. For dissociation curve analysis, the instrument collects data during a pre-programmed temperature ramp and plots the negative of the first derivative for normalized fluorescence over time. Note For detailed information on any of the chemistries supported by the ABI PRISM 7900HT Sequence Detection System, see the appropriate chapter for the type of run (see Chapter 5, “End-Point Analysis,” and Chapter 6, “Real-Time Analysis,” respectively). 2-2 Product Overview Section: Getting to Know the Hardware In This Section This section contains the following information: Topic See Page 7900HT Instrument 2-4 Computer 2-7 Bar Code Readers 2-8 Zymark Twister Microplate Handler 2-9 Compatible Consumables 2-10 Instrument Connections 2-11 GR2009 Instrument The ABI PRISM 7900HT Sequence Detection System consists of the following Components components: 1 Number 1 2 4 4 5 6 For information on the… See Page 7900HT Instrument 2 3 3 2-4 Fixed-Position Bar Code Reader Automation Accessory (optional) Zymark® Twister™ Microplate Handler Extended Capacity Stacks 5 Hand-Held Bar Code Reader 6 Microsoft® Windows®-Compatible 2-8 2-9 2-8 Computer 2-7 Product Overview 2-3 7900HT Instrument Internal The 7900HT instrument contains the hardware used for thermal cycling and detection Components of fluorescent chemistries (see page 2-2). The figure below illustrates the major subcomponents of the instrument described in further detail below. Laser CCD camera Optics system Reader GR2112 Heated clamp Tray Door Automated plate handling system Sample block module ! CAUTION Do not remove the cover to the ABI PRISM 7900HT Sequence Detection System. Only a qualified Applied Biosystems service engineer may repair or adjust the internal components of the 7900HT instrument. Failure to comply can result in serious injury and/or damage to the instrument. Automated Plate ! WARNING PHYSICAL HAZARD. Keep hands and loose clothing away from the instrument Handling System tray and door at all times during instrument operation. The 7900HT instrument contains several internal components that can cause serious physical injury. The 7900HT instrument features an automated plate handling system to provide easy loading and removal of plates from the instrument. In combination with the automation module, the plate handling system allows unattended operation of the instrument. Heated Clamp ! WARNING PHYSICAL INJURY HAZARD. Hot Surface. Use care when working around this area to avoid being burned by hot components. The 7900HT instrument features a heated clamp that provides optimal heat transfer and uniform heating during thermal cycling. When the instrument tray loads a plate, the clamp applies a downward pressure of 70 lbs (31.8 kg) onto the consumable. During the run, the clamp maintains a constant temperature of 105 °C (± 3 °C) to prevent condensation within the consumable wells. 2-4 Product Overview Interchangeable ! WARNING PHYSICAL INJURY HAZARD. Hot Surface. Use care when working around Thermal Cycler this area to avoid being burned by hot components. Blocks The 7900HT instrument features a Peltier-based, interchangeable sample block module based on the technology established in the GeneAmp ® PCR System 9700 thermal cycler. The internal Peltier heating/cooling unit is housed in the sample block module. The sample block module is made of aluminum to provide the optimal thermal transfer rate. Circuitry and connections to the instrument (Do Not Touch) Warning GR2028 GR2028 Heat sinks (Do Not Touch) Top View Bottom View The sample block module provides: ♦ Wide temperature range: 4–99.9 °C ♦ Accuracy: ±0.25 °C from 35–99.9 °C ♦ Heat/cool rate: 1.5 °C per second ♦ Temperature uniformity: ±0.5 °C (measured 30 sec after the clock starts) ♦ Long-term stability and high reliability The interchangeable sample block module… See Page supports multiple consumable formats. 2-10 provides several different modes of operation (including 9600 mode and programmable temperature ramps). 4-13 reduces instrument downtime by allowing immediate replacement of the block. 7-4 permits easy access to the sample block for troubleshooting and maintenance. Product Overview 2-5 Optics System IMPORTANT Do not remove the cover to the 7900HT instrument. Only a qualified Applied Biosystems service engineer may repair or adjust the internal components. The optical system of the 7900HT instrument is based on the optics system found in the ABI PRISM ® 7700 Sequence Detection System. The figure below illustrates the components of the 7900HT optics system. Note For more information about the operation of the optical system of the 7900HT instrument, see Appendix A, “Theory of Operation.” Charged coupled device array Camera lens Grating Emission filter Beam splitter Laser source Fresnel GR2103 Lens (within Lensplate) 384/96-Well optical plate Side View 2-6 Product Overview Front View Computer Description The computer coordinates the operation of the Sequence Detection instrument, automation module, and the bar code readers via the SDS software. The figure below illustrates the general configuration of the computer supplied with the ABI PRISM 7900HT Sequence Detection System. Monitor power button GR2009 Computer power button System IMPORTANT The following requirements are valid for Version 2.0 of the SDS software and Requirements may change with future revisions of the 7900HT instrument software and firmware. Check the release notes accompanying your version of the SDS software for updates. To run the SDS software and/or to operate the ABI PRISM 7900HT Sequence Detection System, a computer must meet the following minimum requirements: Component Minimum Requirement Processor Intel Pentium II processor, 400 MHz or faster Memory 256 MB RAM Hard Drive Minimum 25 GB available hard disk space Additional Drives CD-ROM drive Operating Systems Microsoft Windows NT 4.0 with Service Pack 6A Hard Drive During installation of the 7900HT instrument, the computer hard drive was partitioned Partitions to create the following logical drives: Partition C Size (GB) 2 Contains Operating system files Note Applied Biosystems recommends that you do not install programs to the C drive. The computer will boot faster if the C drive contains only the operating system. D ≥25 ♦ SDS 2.0 Software ♦ Automation Controller Software ♦ Additional Third-Party Software ♦ ABI PRISM SDS Plate Document Files Product Overview 2-7 Bar Code Readers Description The ABI PRISM 7900HT Sequence Detection System incudes two bar code readers for data entry and plate recognition: ♦ a hand-held bar code reader for scanning plates manually ♦ a fixed-position bar code reader for automated scanning of plates as they are loaded into the instrument (available with automation accessory only) Both bar code readers use a 488 nM laser to scan plates and are capable of reading Code 128 (alpha-numeric), which supports 128 ASCII characters. Locations of the The following figure illustrates the locations of the bar code readers in the system. Bar Code Readers 1 Fixed-Position Bar Code Reader GR2009 2 Hand-Held Bar Code Reader 1 2 Splitter To computer keyboard port To keyboard GR2015 GR2018 ~~ ~ ~ ~ ~ (Shown with cover removed) Using the ! WARNING LASER HAZARD. Exposure to direct or reflected laser light can burn the retina Bar Code Readers and leave permanent blind spots. Never look into the laser beam. Remove jewelry and anything else that can reflect the beam into your eyes. Protect others from exposure to the beam. For directions on... 2-8 Product Overview See Page Using the Hand-Held Bar Code Reader 3-19 Aligning the Fixed-Position Bar Code Reader 7-40 Zymark Twister Microplate Handler Description The Zymark Twister Microplate Handler coordinates plate handling for the ABI PRISM 7900HT Sequence Detection System permitting 24-hour unattended operation. The arm features a 310-degree rotational swing that permits access to the 7900HT instrument drawer, up to five plate stacking areas, and the fixed-position bar code reader. To... See Page Turn on the Automation Module 3-5 Align the Automation Module 7-27 Zymark Twister The plate handler consists of the following components: Microplate Handler Components GR2014a Adjustment knob Gripper Plate-sensor switch (cross-sectional view of the gripper) Plate stack Expansion stacks Zymark Twister Microplate Handler Fixed-position bar code reader Product Overview 2-9 Compatible Consumables Consumables for Use The ABI PRISM 7900HT Sequence Detection System can support a variety of with the 7900HT consumable formats through the use of interchangeable sample block modules. Instrument Applied Biosystems offers sample block modules that support the following consumable formats: Consumable Illustration GR2107 ABI PRISM™ 384-Well Reaction Plate GR2108 ABI PRISM™ Optical 96-Well Reaction Plate Consumable See Appendix D, “Kits, Reagents and Consumables,” for a listing of available Requirements consumables, requirements, and purchasing instructions. 2-10 Product Overview Instrument Connections Electrical The diagram below illustrates the electrical connections between the components of Connections the ABI PRISM 7900HT Sequence Detection System. Power A H HI-POT A Power B C D GR2020 Power HI-POT A B C E A F G B C Power D E Power G D C Communications Cable Power Cable The following table lists the communications cables. Cable Connects… To… Communication Computer (Monitor Port) Monitor Comm/Power Computer (Mouse Port) Mouse (not shown) C Serial Computer (Serial Port 1) 7900HT Instrument D Comm/Power Computer (Keyboard Port) Hand-held Bar Code Reader E Communication Computer (Serial Port 2) Plate Handler (Port C) F Ethernet Network Computer (Ethernet Port) Ga Comm/Power Computer (ISA Card 1) Fixed-Position Bar Code Reader H Comm/Power Bar Code Reader Cable Keyboard (not shown) A B Type a. See the figure below. To Lava Card (ISA Card 1) Power supply Bar Code Reader Cable Power G GR2067 Product Overview 2-11 2-12 Product Overview Section: Getting to Know the Software In This Section This section contains the following information: Topic See Page Sequence Detection System Software Components and Features 2-14 Managing Sequence Detection System Data 2-15 Working with SDS Software Files 2-16 Product Overview 2-13 Sequence Detection System Software Components and Features Software The ABI PRISM 7900HT Sequence Detection System uses several software Components applications to set up, run, and analyze experiments completed on the 7900HT instrument. Application Function SDS Software ♦ Constructs and edits plate document files (*.sds files) ♦ Performs initial and end analysis of raw data from allelic discrimination and absolute quantification runs ♦ Saves, prints, and exports run data Automation Controller Software ♦ Controls and coordinates the action of the 7900HT instrument and the automation module ♦ Initiates and controls the sequence detection run ♦ Acquires data during the run Java® Runtime Environment It includes additional files and software used to run the SDS software. IMPORTANT Do not delete the Java Runtime Environment files. These files are crucial to the operation of the SDS software. If the files are deleted or become corrupt, reinstall the SDS software from the CD as explained on page 7-48. Zymark Twister Software Used to calibrate the Zymark Twister Microplate Handler. LAVA Software Used to align the fixed-position bar code reader. Instrument Firmware ♦ Controls the most basic operations of the 7900HT instrument ♦ Controlled by commands sent from the computer ♦ Acts as the link between the software commands and hardware operations 2-14 Product Overview Managing Sequence Detection System Data 7900HT Instrument Data management strategy is a crucial element of successfully integrating the Dataflow ABI PRISM 7900HT Sequence Detection System into a laboratory workflow. During a single 24-hour period of real-time operation, the 7900HT instrument can produce up to 200 MB of data. To manage the information produced by the 7900HT instrument successfully, it helps to have a basic understanding of how data is collected and processed prior to analysis. The figure below contains a summary of the 7900HT instrument data flow. ABI PRISM 7900HT Sequence Detection System Instrument Firmware Thermal Cycling and Sequence Detection Data Collection Serial Cable Computer Automation Controller/ SDS Software ♦ SDS files added to and run from the plate queue Raw data saved to SDS files ♦ SDS file run individually Hard drive *.sds *.sds files files SDS Software Plate document creation Data analysis Downstream application specific analysis Product Overview 2-15 Working with SDS Software Files Files Used and IMPORTANT Never move or delete a SDS software file or folder unless specifically directed to Created by the do so by an Applied Biosystems representative or documentation. SDS Software The SDS software includes many different files and folders. Some of these are created to store run data and calibration data, others are required to run the software. File Type Extension Description Plate Document Files ABI PRISM SDS Single Plate *.sds A plate document is a virtual representation of a consumable (plate) used to contain samples and reagents during an sequence detection run. During the run, the software uses the plate document to coordinate the operation of the instrument (thermal cycling, data collection), to organize and store the data gathered during the PCR, and to store the results of the downstream analysis of the run data. ABI PRISM SDS Template File *.sdt Templates contain can be used to create an unlimited number of plate documents. Templates are optional but useful as a time-saving devices for experiments where samples are run on plates with identical assay configurations. Imported/Exported Files Tab-delimited text file *.txt The SDS software can export raw or analyzed data in tab-delimited (*.txt) format for all or a select group of wells on a plate document. The exported files are compatible with most spreadsheet applications. JPEGa graphic files *.jpg The SDS software can export most panes and plots of the plate document as JPEG graphic files. The JPEG format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software. a. Joint Photographic Experts Group Average SDS Plate The SDS software can produce SDS files of varying sizes depending on the type of Document File Size runs for which they are created. The table below lists the average sizes of typical files produced by the SDS software. Compressed File Sizea Run Type Average File Size Plate-read 150–180 KB 70–90 KB Real-timeb 15–25 MB 10–15 MB a. Compressed files sizes shown are estimates based on standard compression using the WinZip® utility. For more information, see “Archiving SDS Files” on page 7-46. b. The maximum file sizes displayed above are nominal for real-time runs (absolute quantification). File size can increase depending on the plate document’s data collection options. 2-16 Product Overview Getting Started 3 3 In This Chapter This chapter discusses the following topics: Topic See Page Getting Started 3-2 About This Manual 3-3 Turning on the ABI Prism 7900HT Sequence Detection System 3-5 Using the SDS Software Workspace 3-7 Basic Software Skills Tutorial 3-11 Using SDS Plate Documents 3-21 Getting Started 3-1 Getting Started Before You Begin If this is your first time using the ABI PRISM® 7900HT Sequence Detection System, consider completing the “Basic Software Skills Tutorial” on page 3-11 before continuing. The tutorial will provide you with the fundamental knowledge required to operate the SDS software and will teach you time-saving techniques to allow you to use it quickly and efficiently. Procedure The following table contains a list of major procedures described within this manual. Quick Reference Procedure Turning on the ABI PRISM 7900HT Sequence Detection System See Page 3-5 Setting Up and Running SDS Experiments Creating an SDS plate document 4-6 Running an individual SDS plate document 4-21 Running batches of SDS plate documents (using the automation accessory) 4-29 Stopping a run in progress ♦ From the SDS software 4-26 ♦ From the Automation Controller Software 4-38 Ejecting a plate ♦ From the SDS software 4-27 ♦ From the Automation Controller Software 4-38 Analyzing Run Data Analyzing an allelic discrimination run 5-3 Analyzing an absolute quantification run 6-3 Analyzing a dissociation curve (melting curve) run 6-17 Maintaining the 7900HT Instrument Changing the 7900HT Plate Adapter 7-9 Replacing the Sample Block 7-4 Decontaminating the Sample Block 7-11 Performing a Background Run 7-13 Performing a Pure Dye Run 7-17 Adding Custom Dyes to the Pure Dye Set 7-21 Verifying Instrument Performance Using a TaqMan RNase P Plate 7-24 Maintaining the Automation Accessorya Adjusting the Sensitivity of the Plate Sensor Switch 7-28 Aligning the Plate Handler 7-32 Aligning the Fixed-Position Bar Code Reader 7-40 Cleaning and Replacing Gripper Finger Pads 7-43 a. The automation accessory includes the Zymark® Twister™ Microplate Handler and the fixed-position bar code reader. See “Instrument Components” on page 2-3 for more information. 3-2 Getting Started Using the SDS The SDS software features an online help system that can guide you through the Software procedures for setting up, performing, and analyzing runs. To get help at any time, Online Help click a Help button located within the dialog box or window in which you are working. The SDS software provides two ways to access the online help as follows: To… Then… access general help select SDS Online Help from the Help menu. get help for using a specific dialog box, plot, or feature click a help button ( ) located within the dialog box or window in which you are working. For More For information about the ABI PRISM 7900HT Sequence Detection System or the SDS Information software, Applied Biosystems recommends the following references: Title P/N ABI PRISM 7900HT Sequence Detection System Site Preparation and Safety Guide 4317595 ABI PRISM 7900HT Sequence Detection System Software Online Help — Microsoft Windows Operating System Online Help — About This Manual Intended Audience This guide is written for technicians, scientists, and researchers who will use ABI PRISM 7900HT Sequence Detection System (SDS) instruments. Background Needed This manual assumes that you are familiar with the following: ♦ Basic Microsoft® Windows® operations such as using the mouse, choosing commands, working with windows, and using the hierarchical file system ♦ A general understanding of electronic storage devices and data files ♦ An understanding of assay preparation and basic laboratory techniques Computer To use the ABI PRISM 7900HT Sequence Detection System, you should be familiar Vocabulary and with the following basic computer vocabulary and operations: Operations Vocabulary and Operations Description Using the mouse Clicking and double-clicking, selecting, and dragging. Choosing commands Using menus and drop-down lists, dialog boxes, radio buttons, and check boxes. Working with windows Opening and closing, resizing and repositioning, scrolling, understanding the active window. Using the Microsoft Windows hierarchical file system Finding files and creating folders. Getting Started 3-3 Conventions Used in This manual uses the following conventions to convey information: This Manual Convention > Bold text Definition Examples This symbol is used to convey a command or directory path in the Windows operating system. ♦ From the Start menu, select Programs > SDS 2.0 > SDS 2.0. Bold text appearing with procedures corresponds to the text as it appears on the screen. ♦ From the File menu, select Save. ♦ Navigate to the Program Files > Applied Biosystems > SDS 2.0 > Templates directory. ♦ The Detector Manager dialog box opens. How This Manual is This manual contains the following chapters and supporting appendices: Organized 3-4 Getting Started Chapter/Appendix Content 1 Safety Explains information on ABI PRISM 7900HT Sequence Detection System safety 2 Product Overview Describes the components of the ABI PRISM 7900HT Sequence Detection System and its software 3 Getting Started Introduces and explains how to use this manual and the SDS software 4 Run Setup and Basic Operation Explains how to create and run plate documents on the ABI PRISM 7900HT Sequence Detection System 5 End-Point Analysis Describes how to analyze data from allelic discrimination experiments 6 Real-Time Analysis Describes how to analyze data from absolute quantification and dissociation curve experiments 7 System Maintenance Explains how to perform both routine and incidental system maintenance for the components of the ABI PRISM 7900HT Sequence Detection System 8 Troubleshooting Contains tips for troubleshooting problems with the ABI PRISM 7900HT Sequence Detection System 9 User Bulletins This chapter is reserved for user bulletins A Theory of Operation Describes the principles behind the operation of the ABI PRISM 7900HT Sequence Detection System B Importing and Exporting Plate Document Data Explains the Import/Export function of the SDS software and also diagrams the structure and annotation of setup table files C Designing TaqMan Assays Contains brief instructions for designing TaqMan probe and Sequence Detection primer sets D Kits, Reagents and Consumables Contains a list of Applied Biosystems kits and consumables for use with the 7900HT instrument E References Contains a bibliography of references for this manual F Contacting Technical Support How to contact Applied Biosystems Technical Support G Limited Warranty Statement The Applied Biosystems limited warranty statement Turning on the ABI PRISM 7900HT Sequence Detection System Turning On the The activation of the ABI PRISM 7900HT Sequence Detection System is sequential, 7900HT Instrument each component must be activated in a specific order for the system to initialize properly. If performed out of sequence, the components may not be able to establish the necessary communication connections. Monitor power button GR2009 Status lights 7900HT instrument power button Computer power button Zymark Twister Microplate Handler (power button in the rear) IMPORTANT Turn on the power to the instrument and the plate handler at least 10 min before use. When activated, the instrument heats the sample block cover to 105 °C. If a run is started before the heated cover reaches 105 °C, the instrument will pause until it reaches the optimal temperature before commencing the run. To activate the components of the ABI PRISM 7900HT Sequence Detection System: Step Action 1 Turn on the monitor and computer. 2 Turn on the Zymark Twister Microplate Handler by pressing the power switch located on the back panel of the plate handler (see below). ! CAUTION PHYSICAL HAZARD. Keep clear of the arm when activating the plate handler. Once activated, the arm automatically moves to its home position. Power switch HI-POT B C D GR1728 A Rear Panel of the Twister If operating normally, the plate handler moves the arm to the home position (over the output stack). 3 Turn on the 7900HT instrument by pressing the power button located below the status lights on the front of the instrument (see the figure at the top of the page). If operating normally, the 7900HT instrument will do the following on startup: ♦ Emit a high-pitched tone signalling that system has been initialized. ♦ Cycle the status lights (Red > Orange > Green) indicating that the 7900HT instrument is active (see “Reading the Instrument Status Lights” on page 3-6 for more information). Getting Started 3-5 Reading the The 7900HT instrument contains three lights located on the lower-left side of the front Instrument panel to indicate the status of the instrument. Status Lights Red Orange Green Status lights Power button GR2010 Light/Appearance Status Action Green Solid The 7900HT instrument is on and in idle state (ready to run) None Flashing ♦ Interlocks are open and/or the scan head has not reached the safe position. This state indicates normal instrument function. ♦ The instrument door is open. Orange Flashing Solid The 7900HT instrument is transmitting/receiving data to/from the computer (usually during a run). None If the light remains on during startup for more than 2 min: a. Check that the computer is turned on and connected to the instrument. (See page 2-11 for a diagram of instrument connections.) ♦ The instrument did not boot properly, or Red 3-6 Getting Started Solid This state indicates normal instrument function. ♦ 7900HT instrument has experienced a system failure b. If so, turn off the instrument, wait for 30 sec, and then restart as explained on page 3-5. The 7900HT instrument has detected a fatal problem. Turn off the instrument, wait for 30 sec, and then restart. Using the SDS Software Workspace Launching the To launch the SDS software, either: Software ♦ Select Start > Programs > SDS 2.0 > SDS 2.0, or ♦ Double-click the SDS software program icon on the desktop. Select SDS 2.0, or Double- click The computer launches the SDS software and attempts to establish communication with the 7900HT instrument. If the connection is successful, the software displays the Connected icon ( ) in the status bar when a plate document is open. See “About the Status Bar” on page 3-10 for more information. Getting Started 3-7 About the All software operations and displayed information occur within the workspace of the Software Interface SDS software. The workspace provides quick access to all elements of the software through the menubar and a pair of toolbars. The following figure summarizes the features of the user interface of the SDS software. 1 2 3 4 6 5 The following table describes the elements of the workspace: Number Component Description 1 Menubar Contains a directory of menus that govern the operation of the software. 2 General Toolbar Contains clickable icons for controlling the basic functions of the software (file management and basic editorial). 3 Display Toolbar Contains clickable icons for controlling the display of information within the SDS software workspace. 4 Workspace Contains all plate documents, dialog boxes, and message boxes used by the SDS software. 5 Message Bar Displays a variety of messages to indicate the status of the instrument. Note See “About the Status Bar” on page 3-10 for a complete description of the message bar. 6 Instrument Connection Icon Indicates the status of the connection to the 7900HT instrument. Note See “About the Status Bar” on page 3-10 for a complete description of the instrument connection icon. 3-8 Getting Started Using the General The following table describes the icons located within the General Toolbar: Toolbar Icon Function Creates a new plate document Opens an existing plate document Saves the current plate document Imports data from a text file Exports data to a tab-delimited text file Opens the SDS software Find utility Removes the selected object and places it into memory Copies the selected object into memory Inserts a cut object into the current selection Analyzes the current plate document Opens the Analysis Options dialog box for the current plate document Using the Display The following table describes the icons located within the Display Toolbar: Toolbar Icon Function Hides or shows the Well Inspector Panel Hides or shows the Plate Grid Hides or shows the Table View Hides or shows the System Raw Data Plot Hides or shows the Multicomponent Plot Hides or shows the Amplification Plot Hides or shows the Standard Curve Plot Hides or shows the Dissociation Plot Zooms the plate grid in or out Opens the Display Settings dialog box used to modify the appearance of the plate document plate grid, plots, and views Getting Started 3-9 About the Status Bar The status bar consists of two components: a message bar for indicating the status of software functions and a instrument status icon for indicating the status of the instrument. About the Message Bar The message bar displays a variety of messages to indicate the status of the instrument. The following table summarizes all of the messages displayed in the Message bar. Message Then the SDS software is… Ready ready and awaiting instructions. Collecting Data currently running a plate document. Reanalyze data requesting analysis of plate document data. The Analysis Options for the plate document have been changed and the document requires reanalysis for them to take effect. Analyzing data... + Progress bar completing the calculations for the current analysis. The metered bar to the right of the message displays the progress of the analysis. Saving data... + Progress bar saving the plate document or template to a storage device. The metered bar to the right of the message displays the progress of the action. importing a file. Importing data... The metered bar to the right of the message displays the progress of the action. Exporting data... + Progress bar exporting the data within the current plate document to a file. The metered bar to the right of the message displays the progress of the action. About the Instrument Status Icon Indicates the status of the connection to the 7900HT instrument. Icon Instrument Status Connected and awaiting a command Not connected or turned off 3-10 Getting Started Basic Software Skills Tutorial About This Tutorial This tutorial will: ♦ Teach you to create, save, print, export, and import SDS plate documents ♦ Familiarize you with the basic components of the SDS software interface ♦ Explain how to customize and arrange the user interface to suit your needs ♦ Teach you to use the hand-held bar code reader ♦ Provide you with time-saving devices to increase your effectiveness on the SDS software Using the Online Version of the Basic Skills Tutorial The SDS Online Help features a version of this tutorial. If you prefer to follow the online tutorial open the SDS Online Help as follows: Step 1 Action If not already active, launch the SDS software as explained on page 3-7. The SDS software workspace appears. 2 From the Help menu, select SDS Online Help. 3 When the SDS Online Help appears, select Basic Skills Tutorial from the list of options. 4 Follow the directions displayed on your screen. Lesson 1: Using Every plate run on the ABI PRISM 7900HT Sequence Detection System requires the Plate Documents creation of a plate document within the SDS software. A plate document is a virtual representation of a consumable used to contain samples and reagents during a sequence detection run. The software uses the plate document to coordinate the operation of the instrument (thermal cycling and data collection), to organize and store the data gathered during the PCR, and to analyze the run data. The SDS software can produce the two types of plate document files described in the table below. Plate Document File Extension Description ABI PRISM SDS Single Plate *.sds SDS Single Plate Documents are the primary file you will use. They are generated for every kind of experiment and are generally used to run plates. ABI PRISM SDS Template Document *.sdt Although optional, templates are useful as time-saving devices for experiments where samples are run on plates with identical assay configurations. The exercises on the following pages will familiarize you with the use of SDS plate documents. Getting Started 3-11 Exercise 1: Creating a Plate Document You will need to create a plate document for every plate you run on the 7900HT instrument. The following procedure explains how to create a plate document using the SDS software. To create a plate document: Step 1 Action If not already active, launch the SDS software as explained on page 3-7. The SDS software workspace appears. 2 Choose one of the following options: ♦ Click the New Document button ( ) from the General toolbar, or ♦ From the File menu, select New. The New Document dialog appears. IMPORTANT The SDS software can handle multiple documents simultaneously, however the processing speed of your computer will decrease with each open document. For that reason, Applied Biosystems recommends limiting the number of open documents to 10. 3 4 Configure the New Document dialog box with the following settings: Drop-Down List Select… Assay Absolute Quantification Container 384 Wells Clear Plate Template Blank Template Click OK. The software displays a new plate document with appropriate attributes. Exercise 2: Saving a Plate Document The Save command stores any changes to the plate document setup information and display settings. The following procedure explains how to save the open plate document. To save the plate document: Step 1 Action Choose one of the following options: ♦ Click the Save button ( ) from the General toolbar, or ♦ From the File menu, select Save As. 2 From the File of type drop-down list, select ABI PRISM SDS Single Plate (*.sds). 3 Click the File name text field, and type Practice. 4 Click Save. The software saves the plate document to a file entitled Practice.sds. Note 3-12 Getting Started Do not close the plate document at this time. Exercise 3: Opening a Plate Document In this exercise you will be opening a template file that you will use in the following exercises. To open a plate document: Step 1 Action Choose one of the following options: ♦ Click the Open button ( ) from the General toolbar, or ♦ From the File menu, select Open. 2 From the Look In text field of the Open dialog box, navigate to Program Files > Applied Biosystems > SDS 2.0 > Templates. 3 From the File of type drop-down list, select ABI PRISM SDS Template Document (*.sdt). 4 From the Look In text field, click the file entitled ‘384 Well RNaseP Install Plate.sdt’ to select it. 5 Click Open. The software opens the plate document file. Exercise 4: Exporting Data from a Plate Document In the following exercise, you will export the plate setup so that you can import it (see Exercise 6). The SDS software allows you to export several components of the plate document as tab-delimited text files, a format compatible with most spreadsheet applications. Note For more information on exporting setup table data using the SDS software, see Appendix B, “Importing and Exporting Plate Document Data.” To export the contents of the template to a setup table file: Step 1 Action Choose one of the following options: ♦ Click the Export button ( ) from the General toolbar, or ♦ From the File menu, select Export. 2 From the Export drop-down list of the Export dialog box, select Setup Table. 3 Select the All Wells radio button. 4 Click the File name text field, and type Practice. 5 Click Export. The software saves the plate document setup table information to a tab-delimited text file entitled ‘Practice.txt’. Note The software also can export the data from most of the analysis plots, graphs, and tables. See Appendix B, “Importing and Exporting Plate Document Data,” for more information. Getting Started 3-13 Exercise 5: Closing a Plate Document When finished viewing or editing a plate document, you will need to close it. If the plate document has been altered since last saving it, the software will prompt you to save the document. In the following procedure, you will close the template document opened in Exercise 3. To close a plate document: Step Action 1 From the File menu, select Close. 2 If prompted to save the plate document, click No. The SDS software closes the file without saving it. Exercise 6: Importing Setup Table Data into a Plate Document As a time-saving device, the SDS software allows you to import a setup table information into a plate document from an exported tab-delimited text file. To illustrate this feature, import the plate grid setup information contained in the Practice.txt file (from Exercise 4) into the empty plate document created in Exercise 1. To import a setup table file into an empty plate document: Step 1 Action If the plate document from Exercise 1 is still open, go to step 2. Otherwise, create a new plate document to receive the setup table data as follows: a. From the File menu, select New. b. Configure the New Document dialog box with settings for the new template. Drop-Down List Select… Assay Absolute Quantification Container 384 Wells Clear Plate Template Blank Template c. Click OK. The software displays a new plate document with appropriate attributes. 2 Choose one of the following options: ♦ Click the Import button ( ) from the General toolbar, or ♦ From the File menu, select Import. 3 From the Look In text field of the Import dialog box, select the Practice.txt file created in Exercise 4. 4 Click Import. The software imports the setup information of the Practice.txt file into the plate grid and table of the empty plate document. Note For more information on importing and exporting setup table data using the SDS software, see Appendix B, “Importing and Exporting Plate Document Data.” 3-14 Getting Started Lesson 2: Viewing Because plate documents can display setup and analysis data in multiple views and Resizing Panes simultaneously, the SDS software has been designed with several navigational devices to help manage the information. This lesson will teach you to use the different aids to reduce screen clutter and ensure efficient use of the software. Exercise 1: Resizing Panes, Views, and Plots You can resize the panes, views, and plots of plate documents by moving the grey lines dividing them horizontally and vertically. To illustrate the this feature, resize the plate grid pane horizontally as follows: Step 1 Action Click and drag the grey line dividing the plate grid and well inspector to the right. The software expands the plate grid and table pane to the new width. Dividing line (click and drag) 2 Using the grey dividing lines, resize other elements of the plate document until comfortable using the feature. Exercise 2: Maximizing/Minimizing Panes, Views, and Plots You can maximize the panes, views, and plots of plate documents by clicking the sizing buttons embedded within the grey dividing lines. Note Sizing buttons are the arrow-head marks ( ) that appear between adjacent elements of the plate document. When clicked, a sizing button hides the element to which it points. To illustrate the maximize/minimize feature, maximize the plate grid as follows: Step 1 Action Click the down-arrow sizing button ( ) in the divider between the plate grid and the table pane to maximize the plate grid vertically. The software maximizes the plate grid by minimizing the table pane. Sizing button 2 Click and drag the grey divider at the bottom of the plate document to restore the table pane to its original size (using the action described in Exercise 1 above). 3 Using the sizing buttons, maximize/minimize other elements of the plate document until comfortable using the feature. Getting Started 3-15 Exercise 3: Hiding and Showing Panes, Views, and Plots You can also toggle the presence of the plate document panes, views, and plots using the icons in the Display toolbar. To illustrate this feature, hide and show the table pane as follows: Step 1 Action From the Display toolbar, click the Show/Hide Table Pane button ( ). The software removes the table pane from the plate document. 2 Click the Show/Hide Table Pane button ( ) again to show the table pane. The software restores the table pane to the plate document. The display toolbar can be particularly useful for manipulating information shown in the plate document. See “Using the Display Toolbar” on page 3-9 for a list of the other icons of the display toolbar and the panes they control. 3 Practice hiding and showing the other plate document elements by clicking other buttons in the Display toolbar until comfortable using the feature. Lesson 3: Using the The plate grid (see below) is an important interface tool for the SDS software. The Plate Grid software uses the grid to convey information about the plate and allows you to select specific wells for viewing and analysis. The following exercises will teach you how to use and modify the elements of the plate grid. Plate grid Exercise 1: Viewing Well Information The SDS software provides two methods for viewing the information associated with a well or wells of the plate document. To view the information for a well of the plate document, do one of the following: 3-16 Getting Started Action Result Click any well in the plate grid to select it. When selected, the software outlines the well in black and displays the associated information in the well inspector of the Setup tab. Move the mouse pointer over any well of the plate grid. After holding position, the software displays the information for the well in a yellow pop-up window. Exercise 2: Selecting Multiple Wells The SDS software features several methods for selecting wells from the plate grid. The following exercise will familiarize you with most of them. To select groups of wells from the plate grid: Step 1 Action Select a block of wells from the plate grid by doing one of the following: ♦ Click and drag the mouse cursor across the block of wells, or ♦ Click the well at the top-left corner of the block, then while holding-down the Shift key, click the well at the bottom-right corner of the block. The software outlines the selected wells with a black border. 2 Select several isolated wells of the plate grid by doing of the following: a. Hold-down the Ctrl key, and click individual wells to select them. The software outlines the selected wells with a black border. b. While holding down the Ctrl key, de-select wells by clicking individual selected wells. 3 Select an entire column or row of wells using the headers as follows: a. Click the header for row A to select all wells in the row. The software outlines the wells of row A with a black border. b. Press and hold either the Shift or Ctrl key, then click other columns or row headers to select multiple columns. Row A header 4 Select all wells of the plate grid by clicking the top-left corner of the plate grid. The software outlines all of the wells in the plate document with a black border. Button 5 Using the techniques illustrated in steps 1 to 4, practice selecting portions of the plate grid until comfortable using the feature. Getting Started 3-17 Exercise 3: Zooming the Plate Grid You can zoom the plate grid to display the well information by clicking the Zoom Grid button ( ). To illustrate this feature: Step Action 1 Click the Zoom Grid button ( well information. ) and observe how the grid expands to display the 2 Click the Zoom Grid button ( ) again to restore the plate grid to the original size. 3 Practice zooming portions of the plate grid until comfortable using the feature. Exercise 4: Resizing Wells Using the Border Lines You can also adjust the size of the plate grid wells by moving the lines in the row or column headers. To illustrate this feature: Step 1 Action Move the mouse cursor over a border line in the row or column header. The mouse cursor becomes a double arrow ( 2 ). Click and drag the mouse cursor to adjust the well to a new width. The software resizes all wells in the plate grid to match the new width. Mouse cursor 3 3-18 Getting Started Practice resizing the wells of the plate grid until comfortable using the feature. Lesson 4: Using The hand-held bar code reader functions as an extension of the keyboard and can be the Hand-Held used to automatically type bar codes into the SDS software. When the reader is used Bar Code Reader successfully to scan a bar code, it automatically: ♦ transmits the alpha-numeric equivalent of the bar code to the software. The software types the bar code text wherever the cursor is active. ♦ transmits a carriage-return (the equivalent of pressing the Enter key). Exercise: Entering Bar Code Information Using the Hand-Held Bar Code Reader Note The following procedure explains how to enter the bar code number into the plate document from the Document Information dialog box. You can also scan the bar code into the New Document dialog box during plate document creation. To enter a bar code number using the hand-held bar code reader: Step 1 Action From the Tools menu, select Document Information. The Document Information dialog box appears. 2 Click the Barcode text field. Click here The software places the blinking cursor in the text field. 3 While holding the hand-held bar code reader 20 to 30 cm away from a plate, aim at the center of the bar code and press the trigger. The scanner emits a sweeping laser beam that appears as a red line on the plate. Slowly move the scanning beam slowly across the bar code until the scan gun emits a high-pitched tone acknowledging that it has read the code. ! WARNING LASER HAZARD. Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots. Never look into the laser beam. Remove jewelry and anything else that can reflect the beam into your eyes. Protect others from exposure to the beam. PECY001DL3 GR2110 20-30 cm Once the gun has read the bar code, the software automatically populates the selected text field with the alpha-numeric equivalent of the bar code. 4 Click OK to close the Document Information dialog box. Getting Started 3-19 Lesson 5: Using The SDS software features contextual menus as a time-saving devices that provide Contextual Menus access to the commands for an associated view or pane. To access a contextual menu, move the mouse pointer over a pane or view of interest and click the right mouse button. The menu appears at the location of the pointer. Contextual menu All contextual menus provide the following common commands: Command Result See Page Hide <pane or plot> Hides the pane or view. 3-16 Save <pane or plot> to Image File Opens the Export Graphic dialog box for exporting the selected view or pane as a JPEG graphic file. B-8 Display Settings Opens the display settings dialog box that allows you to modify the appearance of the view, pane, or plot. 4-17 Lesson 6: Using The SDS software features keyboard shortcuts for invoking the major functions of the Keyboard Shortcuts software. Exercise: Closing the Plate Document To illustrate the use of a keyboard shortcut, close the plate document as follows: Step Action 1 Simultaneously press the Ctrl and W keys (Ctrl+W). 2 When prompted to save the plate document, click No. Note The SDS software online help contains a complete list of the keyboard shortcuts for the SDS software. To view the list, open the online help as explained below. Lesson 7: Using the The SDS software features an online help system that can guide you through the SDS Software procedures for setting up, performing, and analyzing runs. To get help at any time, Online Help click a Help button located within the dialog box or window in which you are working. The SDS software provides two ways to access the online help as follows: s 3-20 Getting Started To… Then… access general help select SDS Online Help from the Help menu. get help for using a specific dialog box, plot, or feature click a help button ( ) located within the dialog box or window in which you are working. Using SDS Plate Documents Using Multiple The SDS software can handle multiple documents simultaneously, however the Plate Documents processing speed of the computer will decrease with each open document. For that reason, Applied Biosystems recommends limiting the number of open documents to 10. Elements of a This section will familiarize you with the elements of plate documents. The figure Plate Document below illustrates a typical plate document. The following pages describe its components. Plate grid Tabbed pages Well inspector Table pane Plate Grid Each plate document consists of a grid that corresponds to the wells of a reaction device. The grid displays well information depending on the type of plate document. The information displayed within the cells of the grid are determined by the Plate Grid properties settings located within the Display Settings dialog box. Note For more information on configuring the display settings for the plate grid, click a help button ( ). Table Pane The table pane displays the setup and analysis properties for the plate document in a tabular format. The table pane can be exported as a tab-delimited text file for use by a spreadsheet application (see Appendix B, “Importing and Exporting Plate Document Data,” for more information). Getting Started 3-21 Tabbed Pages Plate documents can contain up to six tabbed pages depending on their function: Tab Used to... Setup display well information, and to configure the plate grid with setup information. Instrument program the plate document method, and to run the plate document or send it to the Plate Queue. Raw Data display the raw fluorescence collected from the sequence detection run. Calibration Data display the Background and Pure Spectra calibration data used for the signal normalization and multicomponenting analysis of the current run. Results display analyzed run data. The Analysis tab is visible only in plate documents containing analyzed run data. Dissociation Curve display analyzed dissociation curve data from a programmed ramp. The Dissociation Curve tab is visible only in plate documents containing analyzed data from a real-time run with a programmed ramp. Well Inspector The Well Inspector is used to apply detector and sample information to the wells within the grid pane and to display information from the selected cells in the plate grid. Sample name text field Detector list Passive Reference drop-down list In Use check box Component Description Sample name text field An editable text field that displays the sample name applied to the selected well(s) Note The Sample name text field will display *Mixed* if multiple wells with different sample names are selected. Detector list Lists all available detectors copied to the plate document In Use check box Toggles the activity of the well. If unchecked, the software eliminates the data from the selected well from all analysis procedures. Passive Reference Displays the fluorescent dye used as a passive reference drop-down list Marker Inspector Used only for allelic discrimination runs, the Marker Inspector (shown below) appears as the lower half of the Well Inspector and displays all markers available for the plate. Marker inspector 3-22 Getting Started Run Setup and Basic Operation 4 4 In This Chapter This chapter discusses the following topics: Topic See Page Before You Begin 4-2 Setup Checklists 4-3 Section: Plate Document Setup 4-5 Step 1 – Creating a Plate Document 4-6 Step 2 – Applying Detectors and Markers to the Plate Document 4-7 Step 3 – Configuring the Plate Document with Detector Tasks 4-11 Step 4 – Programming the Plate Document Method 4-13 Step 5 – Saving the Plate Document as a Template 4-17 Step 6 – Creating a Plate Document from the Template 4-18 Step 7 – Applying Sample and Plate Information 4-19 Step 8 – Running the Plate on the 7900HT Instrument 4-20 Section: Running an Individual Plate 4-21 Saving the Plate Document 4-22 Preparing and Running a Single Plate 4-23 Operating the 7900HT Instrument Using the SDS Software 4-25 After the Run 4-27 Section: Running Multiple Plates Using the Automation Controller 4-29 Adding Plate Documents to the Plate Queue for Automated Operation 4-30 Adding a Plate Document to the Plate Queue from the SDS Software 4-31 Creating Plate Documents Using the Template Batch Utility 4-32 Running Plates Using the Automation Controller Software 4-34 Loading Plates onto the Automation Module 4-36 Operating the 7900HT Instrument Using the Automation Controller Software 4-38 After the Run 4-38 Run Setup and Basic Operation 4-1 Before You Begin Background Chapters 5 and 6 include brief discussions of the experiments that can be performed Information using the 7900HT instrument. Before beginning, you may want to review the appropriate chapter for your experiment: Run Type Experiment See Page End-point Allelic Discrimination 5-3 Real-time Absolute Quantification 6-3 Dissociation Curve Analysis 6-17 Getting More The SDS software features an online help system that can guide you through the Information from procedures for setting up, performing, and analyzing runs. To get help at any time, button located within the dialog box or window in which you are working. the Online Help click the Maximizing For end-point applications such as allelic discrimination, the throughput of the Throughput for ABI PRISM® 7900HT Sequence Detection System can be increased by dividing the End-Point Runs workload between the 7900HT instrument and several thermal cyclers. Unlike real-time runs, the 7900HT instrument collects data for end-point runs after the completion of the PCR. Consequently, the thermal cycling of end-point plates can be done elsewhere and then transferred to the 7900HT instrument afterwards for data collection and analysis. IMPORTANT To perform the thermal cycling and the plate read using the 7900HT instrument, run the plate first as a real-time plate document and then again as an allelic discrimination plate document (see “Step 4 – Programming the Plate Document Method” on page 4-13 for the procedure). 4-2 Run Setup and Basic Operation Setup Checklists Experiments/Runs See the appropriate page for the type of experiment or run you want to perform: Performed on the See Page 7900HT Instrument Experiment/Run Absolute Quantification 4-3 Allelic Discrimination 4-4 Background 7-13 Dissociation Curve (Melting Curve) 4-4 Pure Dye (Spectral Calibration) 7-17 RNase P Instrument Performance Verification 7-24 Absolute To create and set up a plate document for an absolute quantification run: Quantification Checklist Done Step Description See Page 1 Create an absolute quantification plate document. 4-6 2a a. Create detectors for the absolute quantification probes. 4-7 b. Copy the detectors to the plate document. 4-8 a. Configure the plate document with detector tasks (NTC, Standard, and Unknown). 4-11 b. Assign quantities to the wells of the plate document that contain standards. 4-12 3a 4 5 a. Program the method for the absolute quantification run. 4-13 b. If performing an assay in which you would like to collect dissociation data, add a temperature ramp to the thermal profile to perform a dissociation curve analysis. 4-16 Choose from the following: If running… Then… a single plate continue to step 7. the first plate in a series of plates with identical assay configurations Save the plate document as an ABI PRISM SDS Template Document as explained on page 4-17. 6 Create a plate document from the template created in step 5. 4-18 7 Configure the document with sample names and plate information. 4-19 8 Prepare and run the absolute quantification plate or plates. 4-20 9 Analyze the run data. 6-3 a. Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab-delimited text file. See “Importing Plate Document Setup Table Files” on page B-2 for more information. Run Setup and Basic Operation 4-3 Allelic To create and set up a plate document for an allelic discrimination run: Discrimination Checklist Done Step Description See Page 1 Create an allelic discrimination plate document. 4-6 2a a. Create detectors for the allelic discrimination probes. 4-7 b. Create a marker for each allelic discrimination probe pairing. 4-9 c. Copy the marker(s) to the plate document. 4-10 3a Assign detector tasks to the wells of the plate document (NTC and Unknown). 4-11 4 If you would like to perform thermal cycling of the allelic discrimination plate on the 7900HT instrument, create a real-time plate document for the plate and program it with the the method for the allelic discrimination run. Otherwise, continue to step 5. 4-13 5 Choose from the following: If running… Then… a single plate continue to step 7. the first plate in a series of plates with identical assay configurations Save the plate document as an ABI PRISM SDS Template Document as explained on page 4-17. 6 Create a plate document from the template created in step 5. 4-18 7 Configure the document with sample names and plate information. 4-19 8 a. Prepare the allelic discrimination plate or plates and perform thermal cycling on a designated thermal cycler. 4-20 b. Run the allelic discrimination plate or plates on the 7900HT instrument. 9 Analyze the run data. 5-3 a. Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab-delimited text file. See “Importing Plate Document Setup Table Files” on page B-2 for more information. Dissociation A dissociation curve analysis is performed as part of a real-time PCR run (absolute (Melting) Curve quantification). To perform a dissociation curve, construct a plate document for Checklist absolute quantification as explained on page 4-3 and configure the method with a temperature ramp as explained on page 4-16. 4-4 Run Setup and Basic Operation Section: Plate Document Setup In This Section This section discusses the following topics: Topic See Page Step 1 – Creating a Plate Document 4-6 Step 2 – Applying Detectors and Markers to the Plate Document 4-7 Step 3 – Configuring the Plate Document with Detector Tasks 4-11 Step 4 – Programming the Plate Document Method 4-13 Step 5 – Saving the Plate Document as a Template 4-17 Step 6 – Creating a Plate Document from the Template 4-18 Step 7 – Applying Sample and Plate Information 4-19 Step 8 – Running the Plate on the 7900HT Instrument 4-20 Quick Review: Note The following table includes a set of abridged procedures for activating the components Turning On the of the ABI PRISM 7900HT Sequence Detection System. For a complete explanation of the 7900HT Instrument procedure, see “Turning on the ABI Prism 7900HT Sequence Detection System” on page 3-5. To activate the ABI PRISM 7900HT Sequence Detection System: Step Appearance Turn on the monitor and computer. Power buttons GR2009 1 Action 2 If using an automation module, turn on the Zymark® Twister™ Microplate Handler. HI-POT 3 B C D GR1728 A Press the power switch located on the back panel of the plate handler. (Rear panel of the Twister) Turn on the 7900HT instrument. Press the power button located on the front panel of the instrument below the LED display. Power button Note For a description of the LED display, see “Reading the Instrument Status Lights” on page 3-6. 4 Power switch GR2010 Launch the SDS 2.0 software by doing one of the following: Select ♦ Select Start > Programs > SDS 2.0 > SDS 2.0. or ♦ Double-click the SDS 2.0 icon from the desktop. SDS 2.0 > SDS 2.0, Double- click The computer launches the software. Run Setup and Basic Operation 4-5 Step 1 – Creating a Plate Document About ABI PRISM Every plate run on the 7900HT instrument requires the creation of a plate document Plate Documents within the SDS software. Plate documents are virtual representations of the consumables (MicroAmp® 384/96-well plates) used to contain samples and reagents during runs. Plate documents contain the following information: ♦ Detector information and arrangement on the plate ♦ Marker information and arrangement on the plate (allelic discrimination only) ♦ Sample information and their arrangement on the plate ♦ Method parameters for the run (absolute quantification only) Creating a Plate To create a plate document: Document Step Action 1 If not already open, launch the SDS software as explained on page 4-5. 2 Choose from the following: ♦ From the File menu, select New, or ♦ From the tool bar, click the New Document button ( 3 ). Configure the New Document dialog box with settings for the run as follows: Drop-Down List Select… Assay the type of assay appropriate for your plate. Note If performing a dissociation curve experiment, select Absolute Quantification from the Assay drop-down list. Container the type of plate you intend to run. Template Blank Template Assay drop-down list Container drop-down list Template drop-down list Barcode text field (Leave blank) Note Because this chapter emphasizes the use of a template file, the Barcode text field is left blank. However, if creating a plate document to run a single plate, scan or type the bar code for the plate into the Barcode text field. 4 Click OK. The software displays a new plate document with the appropriate attributes. 5 4-6 Run Setup and Basic Operation Create and copy detectors (and markers) to the new plate document as described on page 4-7. Step 2 – Applying Detectors and Markers to the Plate Document Creating Detectors Before a plate document can be used to run a plate, it must be configured with detector information for the experiment (and marker information if performing allelic discrimination). A detector is a virtual representation of: a TaqMan® probe in a master mix used for detection of a single target nucleic acid sequence, or the SYBR® Green Double-Stranded DNA Binding Dye 1 used for the detection of double-stranded DNA. Before using the plate document, you must create and apply detectors for all assays present on the plate. To create detectors for the plate document: Step 1 Action From the Tools menu of the SDS software, select Detector Manager. The Detector Manager dialog box appears. 2 Click File, and from the drop-down list select New. 3 Click the Name text field, and type a name for the detector. The Add Detector dialog box appears. IMPORTANT The name of the detector must be unique and should reflect the target locus of the assay (such as GAPDH or RNase P). 4 (Optional) Click the Description text field, and type a brief, 32-character description of the assay. 5 Click the Reporter Dye and Quencher Dye drop-down lists and select the appropriate dyes for the assay. Note If creating a detector for a SYBR Green 1 assay, set the Quencher Dye drop down list to Non Fluorescent. Note If using a custom dye not manufactured by Applied Biosystems, you must create and run a pure dye plate for the custom dye before it can be applied to a detector (see “Adding Custom Dyes to the Pure Dye Set” on page 7-21). 6 Click the Color box, select a color to represent the detector using the Color Picker dialog box, and click OK. 7 (Optional) Click the Notes text field, and type any additional comments for the detector (up to 200 characters). Name text field Description text field Dye drop-down lists Color box Notes text field Creation and Modification date stamps Allelic Discrimination Detector Absolute Quantification Detector Run Setup and Basic Operation 4-7 To create detectors for the plate document: Step 8 (continued) Action Click OK to save the detector and return to the Detector Manager dialog box. The software saves the new detector and displays it in the detector list. 9 Repeat steps 2 to 8 to create detectors for all remaining assays on the plate. Note Click the button for information on the features of the Detector Manager dialog box or to view the procedures for editing, deleting, or searching for detectors. 10 Choose from the following: If constructing a plate document for… Copying and Applying Detectors to the Plate Document Then… absolute quantification copy the detector(s) to the plate document as explained in the procedure below. allelic discrimination create markers to the plate document as explained on page 4-9. IMPORTANT Once copied to the plate document, a detector is no longer linked to the corresponding entry in the Detector Manager. Therefore, if a detector is modified in the Detector Manager after it has been copied to a plate document, the detector must be removed from the plate and copied again to update the plate document with the changes. To copy and assign the detectors to the plate document: Step 1 Action From the Detector Manager dialog box of the SDS software, copy the detectors to the plate document as follows: a. While pressing and holding the Ctrl key, click the detectors you want to apply to the plate document. The software highlights the selected detectors. b. Click Copy to Plate Document. The software adds the detectors to the well inspector of the plate document. 2 Click Done to close the Detector Manager dialog box. 3 From the plate grid, select the wells containing the assay for the first detector. Note For easier selection of plate grid wells, use the Ctrl and Shift keys to select wells individually or in groups. See page 3-16 for more information. 4 Apply detector to the selection by clicking the check box for the detector in the Use column of the well inspector. Use check box Detector added to selected wells of the plate document 5 Repeat steps 3 and 4 to apply the remaining detectors to the plate grid. 6 Configure the plate document with detector tasks as explained on page 4-11. 4-8 Run Setup and Basic Operation Creating an Allelic Allelic discrimination plate documents feature the use of ‘markers’ to aid in organizing Discrimination and applying detectors based on the loci they target. A marker is a pairing of two Marker detectors representing chemical assays designed to amplify different alleles of a common locus. The SDS software uses marker information during data analysis to organize and compare the processed run data. IMPORTANT Allelic discrimination plate documents must contain at least one marker. To create a marker for an allelic discrimination plate document: Step Action 1 If the Detector Manager dialog box is open, click Done to close it. 2 From the Tools menu of the SDS software, select Marker Manager. The Marker Manager dialog box appears. 3 Click Create Marker. The Add Marker dialog box appears. 4 Click the Enter name of new Marker text field, type a name for the new marker, and click OK. The new marker appears within the Markers text field. 5 Apply detectors to the new marker and follows: a. From the Markers text field, click the new marker to select it. The software highlights the selected marker. b. From the Available Detectors text field, click a detector that you want to add to the marker. The software highlights the selected detector. c. Click Add Detector. The software applies the detector to the marker and displays it below the Marker entry. d. Repeat steps b and c for the remaining detector. Detectors assigned to Marker ‘CYP 2C9*2’ 6 If evaluating multiple loci, repeat steps 3-5 to create additional markers as needed. IMPORTANT A marker must be configured with two detectors before it can be applied to a plate document. Note Click the button for information on the features of the Marker Manager dialog box or to view the procedure for deleting markers from the Markers list. 7 Apply the marker(s) to the allelic discrimination plate document as explained in “Copying and Applying Markers to a Plate Document” on page 4-10. Run Setup and Basic Operation 4-9 Copying and IMPORTANT Once copied to the plate document, a marker is no longer linked to the Applying Markers to corresponding entry in the Marker Manager. Therefore, if a marker is modified in the Marker a Plate Document Manager after it has been copied to a plate document, the marker must be removed from the plate and copied again to update the plate document with the changes. To copy and apply markers to the plate document: Step 1 Action From the Marker Manager dialog box of the SDS software, copy the allelic discrimination marker to the plate document as follows: a. Click the marker you want to apply to the plate document. The software highlights the selected marker. b. Click Copy to Plate Document. The software copies the marker and associated detectors to the plate document. c. Repeat steps a and b to copy additional markers to the allelic discrimination plate document as needed. 2 Click Done. The software closes the Marker Manager dialog box. 3 Select the wells containing the assays for a marker you configured in the previous procedure. Note For easier selection of plate grid wells, use the Ctrl and Shift keys to select wells individually or in groups. See page 3-16 for more information. 4 From the marker inspector, click the Use check box of the marker you want to add to the selected wells. The software labels the selected wells with the marker and its detectors. Note The detectors associated with the marker are automatically applied to the selected wells when the marker is placed in Use. Detectors associated with the ‘PDAR CYP 2C9*2’ marker Use check box for the ‘PDAR CYP 2C9*2’ marker (selected) 5 If necessary, repeat steps 3 and 4 to assign additional markers to the plate document. 6 Configure the plate document with detector tasks as explained in “Step 3 – Configuring the Plate Document with Detector Tasks” on page 4-11. 4-10 Run Setup and Basic Operation Step 3 – Configuring the Plate Document with Detector Tasks About Detector The detectors applied to each well of the plate document must be assigned a ‘task’ Tasks that defines their specific purpose or function on the plate. The SDS software uses the detector task assignments to determine how to treat the data produced by the wells when analyzing the run data. Detector tasks vary depending on the type of experiment for which the plate document was created. Applying To apply detector tasks to the detectors of the plate document wells: Detector Tasks Step 1 Action Using the Ctrl and Shift keys, select the wells of the plate grid containing samples for a particular task described in the table below. Experiment Task Apply to… Allelic Discrimination Unknown all detectors of wells containing PCR reagents and test samples. NTC all detectors of negative control wells containing reagents for the PCR, but lacking samples. Unknown all detectors of wells containing PCR reagents and test samples for quantification. Standard the appropriate detectors of wells containing PCR reagents and samples of known quantities. NTC all detectors of negative control wells containing PCR reagents, but lacking samples. Absolute Quantification 2 From the well inspector, click the text field in the Task column for each detector entry, and select the appropriate task from the drop-down list. The SDS software labels all selected wells with the task. Task drop-down list 3 Repeat steps 1 and 2 to apply any remaining tasks to the plate document. 4 Choose from the following options: If constructing a plate document for… absolute quantification Then… assign quantities to the standard wells of the plate document as explained on page 4-12. IMPORTANT The software will disregard data from all Standard wells that have Quantity values of 0. allelic discrimination if necessary, set the passive reference for the plate document as explained page 4-12. Otherwise, continue to “Step 4 – Programming the Plate Document Method” on page 4-13. Run Setup and Basic Operation 4-11 Assigning Quantities to Standards for Absolute Quantification For the SDS software to create a standard curve for quantification of unknown samples, absolute quantification plate documents must contain quantity values for the standards contained on the plate. The software expresses quantity as the number of copies of the target sequence present within an individual well of the plate. IMPORTANT The SDS software excludes from analysis data from Standard wells assigned a Quantity value of 0. To apply quantities to the plate document: Step Action 1 From the plate grid of the SDS software, select the replicate wells containing the first standard in the dilution series. 2 From the well inspector, click the text field in the Quantity column for the appropriate detector, type the number of copies of the target template the replicate wells contain, and press Enter. The software labels the selected standard wells with the specified quantity. Quantity text field 3 Repeat steps 1 and 2 to configure the other sets of replicate standard wells on the plate with the appropriate quantities. When finished, the plate grid should contain a complete set of replicate wells labeled with the Standard task and assigned quantities which the software will use to compute the standard curve for the run. 4 If necessary, set the passive reference for the plate document as explained below. Otherwise, program the method for your run as explained on page 4-13. Setting the If using an Applied Biosystems chemistry, use the default Passive Reference setting Passive Reference (Applied Biosystems chemistries use the ROX™ passive reference molecule). If running a custom chemistry, select a dye to use as a passive reference for the run. Note Applied Biosystems recommends using a passive reference to normalize the signals from the reporter dyes (see page A-6 for more information). To set the passive reference dye for the plate document: Step 1 Action From the Passive Reference drop-down list, select the appropriate reference dye. Passive Reference drop-down list Select the appropriate passive reference 2 4-12 Run Setup and Basic Operation Program the method for the run as explained on page 4-13. Step 4 – Programming the Plate Document Method About SDS Methods During a run, the SDS software controls the instrument based on the instructions encoded within the method of the plate document. Each new plate document (except allelic discrimination) contains a default method that must be edited for the specifics of the experiment. Methods contain the: ♦ Thermal Cycler Conditions ♦ Auto Increment Values ♦ Ramp Rates ♦ Data Collection Options ♦ Reaction Volume Setting To create a method for… Then… absolute quantification program the method as explained on page 4-14. allelic discrimination see page 4-14. dissociation curve analysis Dissociation curves are preformed as part of a real-time PCR run (absolute quantification). Therefore, to perform a dissociation curve analysis do the following: a. Program the method for the absolute quantification experiment as explained on page 4-14. b. Add a temperature ramp to the method for dissociation curve analysis as explained on page 4-16. Run Setup and Basic Operation 4-13 Programming The SDS software is designed to maximize instrument throughput and therefore does Methods for Allelic not provide the option to thermal cycle allelic discrimination plate documents. Discrimination Because allelic discrimination experiments are end-point runs that do not require data collection during the PCR, thermal cycling can be performed on a dedicated thermal cycler and then transferred to the 7900HT instrument for data collection and analysis. If you want to thermal cycle the allelic discrimination plates on… Then… a designated thermal cycler go on to “Step 5 – Saving the Plate Document as a Template” on page 4-17 the 7900HT instrument follow the procedure below. Performing Thermal Cycling of Allelic Discrimination Plates on the 7900HT Instrument To perform the thermal cycling and the plate read using the 7900HT instrument, run the plate first as a real-time plate document and then again as an allelic discrimination plate document as explained below. IMPORTANT Follow the procedure below only if you intend to perform the PCR on the 7900HT instrument. Otherwise, perform the PCR on a dedicated thermal cycler and then transfer the plate to the 7900HT instrument for data collection. To conduct allelic discrimination thermal cycling on the 7900HT instrument: Step Action 1 Launch the SDS software. 2 Create a real-time plate document for absolute quantification as described on page 4-6. Note It is not necessary to configure the plate document with detectors. 3 Program the plate document method with the thermal cycling times and temperatures for your protocol as described in “Programming the Method for Absolute Quantification,” below. 4 Run the plate using the real-time plate document as described on page 4-21. Note Although large, the real-time file may be helpful in diagnosing and troubleshooting the experiment later if the data from the allelic discrimination run produces unexpected results. 5 Go on to “Step 5 – Saving the Plate Document as a Template” on page 4-17. Programming the Note The following procedure describes how to configure only the basic features of the Method for Absolute method: thermal cycler conditions, sample volume, and data collection options. To further Quantification customize the method for the plate document, click the button in the Instrument tab and refer to the online help for instructions on configuring the auto increment and ramp rate values. To create a method for the absolute quantification run: Step Action 1 From the SDS software, click the Instrument tab of the plate document. 2 If necessary, select or de-select the 9600 Emulation check box. Note When the 9600 Emulation check box is checked, the SDS Software reduces the ramp rate of the 7900HT instrument to match that of the ABI PRISM® 7700 Sequence Detection System instrument. 4-14 Run Setup and Basic Operation To create a method for the absolute quantification run: Step 3 (continued) Action Modify the default thermal profile for the method as needed: To… Then… adjust step parameters (time/temp) select a text field value, type a new value, and press Enter. Temperature text field (between 4 and 99.9 °C) Time text field (between 0:01 and 98:59 minutes) add a hold, cycle set, or step a. Click step to the left of the location you want to place the new stage. b. Click Add Cycle or Add Hold. The software inserts the stage into the thermal profile. Note To add a step to a stage, select the step to the left of the location you want to place the step and click Add Step. Selected step New step appears here remove a step a. Click the step you want to remove. The software highlights the selected step. b. Click Delete Step to remove the step from the profile. 4 Configure the data collection options for the method as follows: a. Click the Data Collection tab. b. Click each plateau or ramp within the cycle stage of the thermal profile to place a data collection icon at each step. Data collection icons 5 Click the Sample Volume (µL) text field and type the volume of the reactions to be run on the plate. Note ‘Sample Volume’ refers to the entire contents of any well, including buffer blank, or any combination of master mix and nucleic acids. IMPORTANT All wells on one plate must contain the same reaction volume. 6 Choose from the following options: If performing an… Then… absolute quantification run only go to “Step 5 – Saving the Plate Document as a Template” on page 4-17. absolute quantification run with a dissociation curve add a temperature ramp to generate dissociation curve data as explained on page 4-16. Run Setup and Basic Operation 4-15 Programming a Temperature Ramp for Dissociation Curve Analysis To generate the data required to perform a dissociation curve analysis, the 7900HT instrument must be programmed to run a ‘temperature ramp’ in which it slowly elevates the temperature of the samples while collecting fluorescence measurements once every 7-10 seconds (see page 6-18 for a detailed explanation). To add a temperature ramp to the method for dissociation curve analysis: Step 1 Action From the Instrument tab of the plate document, click the Thermal Profile tab. The software displays the Thermal Profile tabbed page. 2 Click Add Dissociation Stage. The SDS software inserts a temperature ramp at the end of the thermal profile consisting of a set of default steps. The default temperature ramp can be customized, however Applied Biosystems recommends the following guidelines to ensure the greatest separation of the derivative peaks during the analysis, and therefore the maximum resolution for the run. Guideline Example The Start and End steps of the temperature ramp must: ♦ be separated by a minimum temperature difference of 35 °C. End step ♦ elapse 15 seconds (0:15) each. Start step The ramp rate setting for the End step of the temperature ramp must be 2 %. End step ramp rate setting A data collection icon must be placed on the temperature ramp in the thermal profile. 3 4-16 Run Setup and Basic Operation Data collection icon Go to “Step 5 – Saving the Plate Document as a Template” on page 4-17. Step 5 – Saving the Plate Document as a Template Adjusting the Because plate documents created from the template will retain its display settings, Display Settings configure the display settings of the template as you would like the child plate (Optional) documents to be displayed. To configure the display settings for the template: Step 1 Action From the View menu of the SDS software, select Display Settings. The Display Settings dialog box appears. 2 Configure the display settings for the Results Grid, and the Results Table. For more information on the Display Settings dialog box or to view the procedures for configuring the display settings for the template, click the button to open the SDS software online help. 3 When finished, click OK. The SDS software applies the new display settings to the plate document. 4 Go to “Saving the Plate Document as a Template,” below. Saving the Plate IMPORTANT Saving the plate document as a template is an optional step and recommended Document as a for instances where the document can be used to create duplicate plate documents for a series Template of plates with identical assay configurations. If you choose not to use your plate document as a template, go to “Step 7 – Applying Sample and Plate Information” on page 4-19. To save the template file: Step 1 Action From the File menu of the SDS software, select Save As. The Save As dialog box appears. 2 From the Look in text field, navigate to the Program Files > Applied Biosystems > SDS 2.0 > Templates directory. Note By saving the template file to the Templates directory, it becomes available from the Template drop-down list in the New Document dialog box. 3 From the File of type drop-down list, select ABI PRISM SDS Template Document (*.sdt). 4 Click the File name text field, and type a name for the template. 5 Click Save. The software saves the template plate document file. 6 From the File menu, select Close. If the software prompts you to save the plate document, click No. The SDS software closes the template file. 7 Create a plate document from the template as explained on page 4-18. Run Setup and Basic Operation 4-17 Step 6 – Creating a Plate Document from the Template Options for Creating The SDS software offers two options for creating plate documents from a template file: Plate Documents individually or in batches. from the Template Option Description See Page Create an individual plate document from the template The procedure below explains how to create a single plate document from a template file for running a plate. By repeating the procedure, you can create as many plate documents as needed. Follow the procedure below. Create multiple plate documents using the Template Batch utility As a faster alternative to the option above, the software includes a Template Batch utility that can simultaneously create multiple plate documents from the template file. 4-32 Creating a Single To create a plate document from the template file: Plate Document Step Action from a Template 1 From the File menu of the SDS software, select New. The New Document dialog box appears. 2 Configure the New Document dialog box as follows: Drop-Down List Select… Assay the same assay as the template. Container the same plate type as the template. Templatea the template file (*.sdt) created on page 4-17. If the template file does not appear within the Template drop-down list, select the file as explained below: a. Click Browse. The Open dialog box appears. b. From the Look in text field, navigate to and select the template file (*.sdt) created on page 4-17. c. Click Open. The Template drop-down list displays the template file. a. The Template drop-down list displays all template files contained in the Templates subdirectory of the SDS 2.0 program directory. 3 (Optional) Click the Barcode text field and either: ♦ Type the bar code number for the plate, or ♦ Scan the plate bar code using the hand-held bar code scanner. 4 Click OK. The software creates a new plate document from the template file. 5 4-18 Run Setup and Basic Operation Configure the new plate document with sample and plate information as explained on page 4-19. Step 7 – Applying Sample and Plate Information Applying Sample The plate document must contain sample attributes to effectively organize and Names to the Plate analyze data produced from the run. Once applied, the software displays the sample Document names within the plate grid and table views. Note You can apply sample names after the plate has been run, but they must be added prior to the analysis of the run data. Note The SDS software features the ability to import setup table information (detector, detector task, and sample name layouts) into a plate document from a tab-delimited text file. See “Importing Plate Document Setup Table Files” on page B-2 for more information. Applying sample names to the plate document: Step Action 1 From the plate grid of the plate document, select the wells containing the first sample. 2 Click the Sample Name text field, type a name for the sample, and press Enter. The software labels the selected wells with the new sample name. 3 Repeat steps 1 and 2 for all remaining samples. Sample Name text field Name appears here 4 Configure the plate document with plate information as explained below. Configuring the To add or edit any plate information (Barcode, Operator setting, or Plate Comments): Plate Document Step Action Information 1 From the Tools menu of the SDS software, select Document Information. (Optional) The Document Information dialog box appears. 2 Edit the Barcode, Operator, or Plate Comments information. Note For more information on the features of the Document Information dialog box or the data it contains, click the button to open the contextual online help. 3 When finished, click OK. 4 Run the plate document and associated plate as explained on page 4-20. Run Setup and Basic Operation 4-19 Step 8 – Running the Plate on the 7900HT Instrument Options for The 7900HT instrument can run prepared microplates individually or in groups using Running SDS Plates the Zymark Twister Microplate Handler. IMPORTANT If you are not using a Zymark Twister Microplate Handler, you must run plates individually. Choose one of the following options to run the plate: Option Description Individual Operation Run the plate individually from the SDS software. 4-22 Automated Operation Run the plate with others as part of a batch from the Automation Controller Software using the Zymark Twister Microplate Handler. 4-31 IMPORTANT You must have a Zymark Twister Microplate Handler to run plates using this option. 4-20 Run Setup and Basic Operation See Page Section: Running an Individual Plate In This Section This section discusses the following topics: Topic See Page Saving the Plate Document 4-22 Preparing and Running a Single Plate 4-23 Operating the 7900HT Instrument Using the SDS Software 4-25 After the Run 4-27 Run Setup and Basic Operation 4-21 Saving the Plate Document Saving the Plate Before the plate document can be run, you must save it as an ABI PRISM SDS Single Document for Single Plate (*.sds) file. Plate Operation To save the plate document: Step 1 Action From the File menu of the SDS software, select Save As. The Save As dialog box appears. 2 3 From the Look in text field, navigate to and select a directory for the software to receive the new file. Click the File name text field and either: ♦ Type a file name for the plate document file, or ♦ Type or scan the bar code number for the plate into the text field. Note The SDS software does not require that the file name match the bar code of the corresponding plate. 4 Click Save. The software saves the plate document to the specified directory. 5 4-22 Run Setup and Basic Operation Run the plate document and associate plate as explained on page 4-23. Preparing and Running a Single Plate Pre-Run Checklist The following tasks must be complete to run a plate on the 7900HT instrument. Done Check See Page A background run has been performed in the last month 7-13 A pure dye run has been performed in the 6 months 7-17 The instrument tray does not contain a plate 4-27 IMPORTANT The instrument tray must be empty to begin a run. If the instrument tray contains a plate, eject and remove it before continuing. Plate Requirements See “Consumables and Disposables” on page D-3 for a complete list of ABI PRISM consumables and ordering instructions. Preparing the Plate To prepare the plate: Step 1 Action Prepare the reactions in an ABI PRISM® Optical Reaction Plate by aliquoting reagents, enzyme, and samples to the appropriate wells of an optical plate. IMPORTANT The arrangement of the reactions (samples and assays) on the plate must match the configuration of the corresponding plate document. 2 Seal the ABI PRISM Optical Reaction Plate with an ABI PRISM® Optical Adhesive Cover or ABI PRISM® Optical Flat Cap Strips. 3 Briefly centrifuge the plate to collect the reactions at the bottom of the wells and to eliminate any air bubbles that may be present. 4 Choose from the following: Experiment Then… Absolute Quantification/ Dissociation Curve Analysis Run the plate as explained in “Running the Plate” on page 4-24. Allelic Discrimination a. Load the plate onto a designated thermal cycler and perform the PCR. b. Briefly centrifuge the plate to draw the reactions to the bottom of the wells and to eliminate any air bubbles that may have formed during thermal cycling. c. Run the plate as explained in “Running the Plate” on page 4-24. Run Setup and Basic Operation 4-23 Running the Plate To begin the single plate run: Step 1 Action From the SDS software, click the Instrument tab of the plate document. The software displays the Instrument tabbed page. 2 From the Real-Time or Plate-Read tab, click Open/Close. The instrument tray rotates to the OUT position. 3 Place a plate into the instrument tray as shown below. Before loading the plate onto the instrument tray, make sure that: ♦ The associated plate document is open in the SDS software. ♦ The plate has been sealed using an optical adhesive cover. Well A1 PECY001DL3 GR2111 IMPORTANT The A1 position is located in the top-left side of the instrument. 4 Do one of the following: ♦ If performing a real-time run, click Start. ♦ If performing a end-point run, click Plate Read. The instrument tray rotates to the IN position and the instrument performs the run or plate-read. Note Before starting the run, the instrument may pause (up to 15 minutes) to heat the heated cover to the appropriate temperature. Note For more information on the elements of the Real-Time and Plate-Read tabs, click the button and see the SDS software online help. The following options are available during and after the completion of the run: To… See Page Monitor the progress of the run 4-25 Stop the run 4-26 IMPORTANT If you must stop a run in-progress for any reason, carefully read the instructions on page 4-26 before halting the run. 4-24 Run Setup and Basic Operation Open the instrument tray (after the run) 4-27 Analyze the run data after the run is complete 4-27 Operating the 7900HT Instrument Using the SDS Software Monitoring The SDS software displays instrument status and run progress in the Real-Time tab Instrument Progress (real-time runs) or the Plate Read tab (end-point runs) of the respective plate document. The following figure shows examples of the tabs during operation of the 7900HT instrument. Real-Time Tab (Real-Time Runs) Report Plate Read Tab (End-Point Runs) Displays… Status the condition of the 7900HT instrument Time Remaining the calculated time remaining in the run Temperature group box (Real-Time Plate Documents Only) Block the actual temperature of the sample block module Cover the actual temperature of the heated cover Sample the calculated temperature of the samples Cycle group box (Real-Time Plate Documents Only) Rep the current cycle repetition Stage the current stage of the thermal cycling State the current condition of the cycle stage Step the current step being run Time the calculated time remaining in the current step Data Collection Stamp group box (End-Point Plate Documents Only) Post the date that the post-reada was performed. a. A ‘post’ read is a plate read performed after a plate has undergone thermal cycling. Note For more information on the elements of the Real-Time and Plate-Read tabs, click the button and see the SDS software online help. Run Setup and Basic Operation 4-25 Stopping the IMPORTANT Read the following directions carefully before stopping a run-in-progress. Sequence Detector Action from the Run Type From the Plate-Read tab of the plate document, click Stop. SDS Software End-Point Runs ♦ Allelic Discrimination Real-Time Runs Has the Cover reading in the Real-Time tab reached 104 °C? ♦ Absolute Quantification ♦ NO (the heated cover temperature is below 104 °C) ♦ Melting Curve ♦ YES (the heated cover temperature has reached 104 °C) From the Real-Time tab of the plate document, click Stop. The instrument has begun to run the plate and is in the process of thermal cycling. Determine a course of action from the options in the following table: Reason for Stopping the Run Then… Forgot to make a change to the plate document such as adding a detector, detector task, or sample name (not including mistakes to the temperature profile) do not stop the run. ♦ Forgot to add a reagent to the plate (such as enzyme or master mix) a. From the Real-Time tab of the plate document, click Stop. ♦ Programmed the plate document with the wrong thermal profile b. Determine how far into the run the instrument has progressed. Allow the instrument complete the run, then edit the plate document before analyzing the data. The software does not use detector or sample information until the plate document is analyzed. c. Based on the state of the run, determine whether the plate can be re-run. d. If necessary, eject the plate and add the missing reaction component. e. If desired, re-run the plate by re-creating the plate document. 4-26 Run Setup and Basic Operation After the Run Analyzing the The run can be analyzed immediately following the completion of the run: Run Data To analyze data from a plate containing assays for… See Page Allelic Discrimination 5-3 Absolute Quantification 6-3 Dissociation Curve Analysis 6-17 Ejecting a Plate The instrument tray must be opened from the plate document that is connected to the (Opening/Closing 7900HT instrument. View the instrument status icon to determine the plate document the Instrument Tray) to use to eject the plate. Instrument Status Action a. From the Window menu of the SDS software, select the plate document connected to the instrument. b. From the plate document, select the Instrument tab c. Click the Plate-Read or Real-Time tab. d. Click Open/Close. a. From the File menu of the SDS software, select New. b. Click OK. c. Click the Instrument tab. d. Click the Plate-Read or Real-Time tab. e. Click Open/Close. Run Setup and Basic Operation 4-27 Disconnecting the The SDS software has the ability to halt all communications with the 7900HT Software from the instrument. The ‘disconnect’ option is designed to permit the simultaneous operation Instrument of both the SDS software and the Automation Controller Software. Because both programs control the ABI PRISM 7900HT Sequence Detection System, one program must relinquish control of the 7900HT instrument before the other can be used to operate it. To… Then… disconnect the software from the instrument a. From the SDS software, click the Instrument tab of the open plate document. b. Click the Real-Time or Plate-Read tab. c. Click Disconnect. Note Once disconnected, the software neither monitors nor controls the 7900HT instrument. reconnect the software (once disconnected) to reconnect to an open plate document: a. From the File menu, select Close to close the plate document. b. From the File menu, select Open. c. From the Look In text field, navigate to and select the plate document of interest. d. Click Open, Upon opening the plate document, the software re-establishes the 7900HT instrument connection. to reconnect to an new plate document, select New from the File menu. Upon creation of the plate document, the software re-establishes the connection with the 7900HT instrument. 4-28 Run Setup and Basic Operation Section: Running Multiple Plates Using the Automation Controller In This Section This section discusses the following topics: Topic See Page Adding Plate Documents to the Plate Queue for Automated Operation 4-30 Adding a Plate Document to the Plate Queue from the SDS Software 4-31 Creating Plate Documents Using the Template Batch Utility 4-32 Running Plates Using the Automation Controller Software 4-34 Loading Plates onto the Automation Module 4-36 Operating the 7900HT Instrument Using the Automation Controller Software 4-38 After the Run 4-38 Run Setup and Basic Operation 4-29 Adding Plate Documents to the Plate Queue for Automated Operation Automation The first step in configuring the ABI PRISM 7900HT Sequence Detection System for Operation and the automated operation, is to add plate documents to the plate queue. The plate queue is Plate Queue a list of plate document files that the Automation Controller Software uses to identify and run associated plates during automated operation. By adding plate documents to the queue, they automatically become available for use with the Zymark Twister Microplate Handler. IMPORTANT Once a plate document has been added to the plate queue, the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue. The ABI PRISM 7900HT Sequence Detection System features several options for adding plate documents to the plate queue. Review the options discussed in the following table and choose the method that best suits your needs: Option See Page Add a plate document to the plate queue from the SDS software. 4-31 Using the Template Batch utility, create batches of plate documents from a template file and add them to the plate queue. 4-32 Add or remove individual or multiple plate documents to the plate queue using the Automation Controller Software. 4-34 4-30 Run Setup and Basic Operation Adding a Plate Document to the Plate Queue from the SDS Software Adding the Plate IMPORTANT A plate document must contain a bar code before it can be added to the plate Document to the queue. See page 4-19 for more information on configuring a plate document with bar code Plate Queue information. To add the plate document to the plate queue from the SDS software: Step 1 Action From the SDS software, send the plate document to the queue as follows: a. Click the Instrument tab. The software displays the contents of the Instrument tabbed page. b. Click the Queue tab. The software displays the contents of the Queue tabbed page. 2 Click Send to Queue. If… Then… the plate document was not saved previously the Save As dialog box appears. a. From the Look in text field, navigate to a directory for the software to save the new file. b. From the Files of type drop-down list, select ABI PRISM SDS Single Plate (*.sds). c. Click the File name text field and either: – Type a name for the plate document file, or – Type or scan the bar code number of the plate into the text field. d. Click Save. The software saves the plate document. the plate document was saved previously 3 the software automatically saves the plate document. When prompted, click Yes to submit the document to the plate queue. Once a plate document has been added to the plate queue, the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue. Note To release the plate document from the queue, launch the Automation Controller Software and remove the plate document from the queue as explained on page 4-35. 4 Click OK to close the dialog confirming that the plate document has been added to the plate queue. 5 From the File menu, select Close. The SDS software closes the plate document. 6 Repeat the procedures in this chapter to create and add additional plates to the queue as needed. 7 When finished creating plate documents, run the enqueued plates as explained in “Running Plates Using the Automation Controller Software” on page 4-34. Run Setup and Basic Operation 4-31 Creating Plate Documents Using the Template Batch Utility About the Template The Template Batch utility allows you to quickly create multiple plate documents from Batch Utility a single ABI PRISM SDS Template file (*.sdt). The Template Batch utility can be a useful time-saving device in situations where samples are run on plates with identical assay configurations. IMPORTANT Plate documents created by the Template Batch utility do not contain sample or plate information. This information must be applied to each plate document individually after the file is run. Generating Plate Note For more information on the elements of the Template Batch dialog box or to view the button and see the SDS software Documents From a procedures for importing or editing Plate IDs, click the online help. Template To create plate documents from a template using the Template Batch Utility: Step 1 Action From the SDS software, open a template file as follows: a. From the File menu, select Open. b. From the File Type drop-down list, select ABI PRISM SDS Template Document (*.sdt). c. From the Look in text field, navigate to and select the template file. d. Click Open. The SDS software displays the template file. 2 Send the plate document to the queue as follows: a. Click the Instrument tab. The software displays the contents of the Instrument tabbed page. b. Click the Queue tab. The software displays the contents of the Queue tabbed page. 3 Click Send to Queue. The Template Batch dialog box appears. 4 Configure the Template Batch dialog box with Plate IDs as follows: a. Click Add Plates. The Add Plates dialog box appears. b. Click the Plate ID text field, and scan the bar code of the first plate in the batch using the hand-held bar code scanner. ! WARNING LASER HAZARD. Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots. Never look into the laser beam. Remove jewelry and anything else that can reflect the beam into your eyes. Protect others from exposure to the beam. c. Repeat step b for every plate in the batch. d. When finished, click Done. The plate bar codes appear within the Plate ID field. 4-32 Run Setup and Basic Operation To create plate documents from a template using the Template Batch Utility: Step 5 (continued) Action Select a destination directory to store the new plate documents as follows: a. Click Browse. b. From the Look in text field, navigate to and select the directory you want to use to receive the new files. c. Click Open. The Template Batch dialog box displays the selected destination directory in the Plate Directory text field. Destination directory 6 Click Create. The software creates plate documents for all entries listed in the Plate ID list, saves them to the destination directory, adds them to the plate queue, and displays a message indicating the number of plate documents it created and sent to the plate queue. 7 Click OK to close the message box. 8 Click Done. The Template Batch dialog box closes. 9 From the File menu, select Close. The SDS software closes the template document. 10 Repeat the procedures in this chapter to create and add additional plates to the queue as needed. Note Once a plate document has been added to the plate queue, the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue. 11 When finished creating and adding plate documents to the queue, run the queue as explained in “Running Plates Using the Automation Controller Software” on page 4-34. Run Setup and Basic Operation 4-33 Running Plates Using the Automation Controller Software Launching the The ABI PRISM 7900HT Sequence Detection System employs the Automation Automation Controller Software for automated operation of the 7900HT instrument. The software Controller Software coordinates the action of the 7900HT instrument, the bar code reader, and the plate handler while acquiring and saving raw data during each run. To launch the Automation Controller Software as follows: Step 1 Action If running the SDS software, do one of the following: ♦ From the Instrument menu, select Disconnect to discontinue communication between the software and the instrument, or ♦ From the File menu, select Exit to close the SDS software. 2 Do one of the following: ♦ Double click the Automation Controller Software icon ( ) on the desktop. ♦ From the Start menu, select Programs > SDS 2.0 > Automation Controller 2.0. The Automation Controller Software launches and displays the main screen. 3 Verify that Plate Queue text field contains plate documents for all plates you intend to run. ♦ To add a plate document to the plate queue, see “Adding Plates to the Plate Queue” on page 4-35. ♦ To remove a plate document from the plate queue, see “Removing Plate Documents from the Plate Queue” on page 4-35. Plate Queue (must contain plate documents for all plates to be run) 4 4-34 Run Setup and Basic Operation Prepare and load the plates onto the plate handler as explained on page 4-36. Adding Plates to the To add plate documents to the plate queue: Plate Queue Step 1 Action From the File menu of the Automation Controller Software, select Add Plates. The software displays the Open dialog box. 2 From the Look in text field, navigate to the directory containing the file or files of interest. 3 While pressing and holding the Ctrl key, click the plate document file(s) to add to the plate queue. The software highlights selected files. IMPORTANT A plate document must contain a bar code before it can be added to the plate queue. See page 4-19 for more information on configuring a plate document with bar code information. 4 Click Open. The Automation Controller Software adds the plate document(s) to the Plate Queue. Note Once a plate document has been added to the plate queue, the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue. Removing Plate To remove plate documents from the plate queue: Documents from the Then… Plate Queue To remove… specific plate documents from the plate queue a. While pressing and holding the Ctrl key, click the plate document files to remove. The software highlights selected files. b. From the File menu, select Clear Selected Plate(s). The software removes the selected plate documents from the plate queue. all plate documents from the plate queue From the File menu of the Automation Controller Software, select Clear All Plates. The software removes all plate documents from the plate queue. Run Setup and Basic Operation 4-35 Loading Plates onto the Automation Module Pre-Run Checklist The following tasks must be complete to run plates on the 7900HT instrument. See the associated page number for details on each procedure. Done Check See Page A background run has been performed in the last month 7-13 A pure dye run has been performed in the 6 months 7-17 The plate queue contains plate documents for all plates to be run 4-6 The instrument tray does not contain a plate 4-38 IMPORTANT The instrument tray must be empty to begin a run. If the instrument tray contains a plate, eject and remove it before continuing. The output stack does not contain plates. – Plate Requirements See “Consumables and Disposables” on page D-3 for a complete list of ABI PRISM consumables and ordering instructions. Guidelines Observe the following guidelines when loading plates onto the plate handler: ♦ Before loading plates onto the plate handler, make sure that for each plate: – the associated plate document has been added to the plate queue – the plate has been sealed using an optical adhesive cover. ♦ Load the plates into the plate handler stacks in any order. The software reads the bar code of each plate before it is run and matches the plate document and method with the plate. The Automation Controller Software can run batches of up to 84 plates in a single session (21 plates/stack). ♦ Orient the plates within the stacks so that well A1 ( ) of each plate corresponds to the locations shown in the illustration below. ♦ Do not place plates in the output stack (X). The Zymark arm uses the empty stack to store used plates after they are run. X 4 3 2 1 Bar code Zymark Twister Microplate Handler (top view) 4-36 Run Setup and Basic Operation Well A1 Loading Plates To load plates onto the automation module: Step Action 1 Following the guidelines on page 4-36, load the sealed plates into the plate handler stacks. 2 From the Automation Controller Software, select the check boxes for the plate stacks containing plates. IMPORTANT If you are not using stack 1 or the Restack option explained below, remove all plates from stack 1 before starting the queue. Under these settings, the plate handler will attempt to stack the run plates from stack 2 in the stack 1 position. If stack 1 contains plates, these settings will cause the plate handler to stop the run. Plate stack check boxes 3 If you want to retain the location of the plates in the stacks on the plate handler, select the Restack when finished check box. When selected, the Restack function instructs the arm to replace a stack of used plates to their original stack and in their original order after the stack has been run. If the option is not selected, the arm will place each group of used plates within the next vacant stack in clockwise order beginning with the Output stack. Note Restacking plates adds significant operating time when running multiple plates. Use the Restack function only when absolutely necessary. 4 Begin the plate queue as explained on page 4-38. Run Setup and Basic Operation 4-37 Operating the 7900HT Instrument Using the Automation Controller Software Starting the Once all plate documents have been loaded into the Plate Queue and the Instrument Plate Queue Control options are configured you may start the plate queue. To begin the plate queue, click Start Queue from the Plate Queue tabbed page. The ABI PRISM 7900HT Sequence Detection System loads the first plate, scans the bar code, and the begins the run. Note Before starting a real-time run, the instrument may pause (up to 15 minutes) to heat the heated cover to the appropriate temperature. Monitoring The Automation Controller Software displays the progress of the current run in the Instrument Progress Thermal Status tab. See page 4-25 for an explanation of the Thermal Status tab. Stopping the To stop the plate queue, click the Stop button on the Automation Controller Software Sequence Detector dialog at any time. from the Automation Then the instrument... Controller Software If the Stop button is clicked... while the plate handler is handling a plate aborts the run and moves the plate handler to the home position. after a plate has been loaded into the instrument, but before the run has started. aborts the current run, ejects the plate, and moves the plate handler to the home position. after the 7900HT instrument has started a run. aborts the current run, ejects the plate, and moves the plate handler to the home position. IMPORTANT Stopping a run during thermal cycling can affect the chemistry of the reactions within the plate. Before stopping a run, carefully read the guidelines on page 4-26 to determine the best course of action. Ejecting a Plate To eject a plate following a halted run, click Open/Close from the Automation (Opening/Closing Controller Software window. the Instrument Tray) After the Run Analyzing the The run can be analyzed immediately following the completion of the run: Run Data To analyze data from a plate containing assays for… See Page Allelic Discrimination 5-3 Absolute Quantification 6-3 Dissociation Curve Analysis 6-17 4-38 Run Setup and Basic Operation End-Point Analysis 5 5 In This Chapter This chapter discusses the following topics: Topic See Page End-Point Runs on the 7900HT Instrument 5-2 Section: Allelic Discrimination 5-3 Overview 5-4 Before You Begin 5-7 Analysis Checklist 5-8 Analyzing a Completed Allelic Discrimination Run 5-9 Calling and Scrutinizing Allelic Discrimination Data 5-10 After the Analysis 5-15 End-Point Analysis 5-1 End-Point Runs on the 7900HT Instrument End-Point Runs End-point is the term used to describe the category of sequence detection runs in which the ABI PRISM® 7900HT Sequence Detection System is used to measure the fluorescence of a biological sample after it has undergone thermal cycling. Unlike real-time runs that can yield quantitative measurements, the focus of end-point experiments is typically to produce a qualitative result. End-point analysis is commonly used in combination with TaqMan® chemistry to confirm the presence or absence of specific target nucleic acid sequence in cells, tissues, or fluid samples. Currently, the SDS software supports one type of end-point analysis: Allelic Discrimination. 5-2 End-Point Analysis Section: Allelic Discrimination In This Section This section contains the following information: Topic See Page Overview 5-4 Before You Begin 5-7 Analysis Checklist 5-8 Analyzing a Completed Allelic Discrimination Run 5-9 Calling and Scrutinizing Allelic Discrimination Data 5-10 After the Analysis 5-15 End-Point Analysis 5-3 Overview Allelic Discrimination on the 7900HT Instrument The ABI PRISM 7900HT Sequence Detection System supports allelic discrimination using TaqMan® probes. Allelic discrimination is the process by which two variants of a single nucleic acid sequence are detected in a prepared sample. Allelic discrimination chemistry can be used for single-nucleotide polymorphism (SNP) detection. Employing the Allelic discrimination on the 7900HT instrument is made possible through the use of 5´ Nuclease Assay for the fluorogenic 5´ nuclease assay (see page A-2). During the PCR, the fluorogenic Allelic Discrimination probes anneal specifically to complementary sequences between the forward and reverse primer sites on the template DNA. Then during extension, AmpliTaq Gold ® DNA polymerase cleaves the probes hybridized to the matching allele sequence(s) present in each sample. The cleavage of each matched probe separates the reporter dye from the quencher dye, which results in increased fluorescence by the reporter. After thermal cycling, the plate is run on the 7900HT instrument which reads the fluorescence generated during the PCR amplification. By quantifying and comparing the fluorescent signals using the SDS software, it is possible to determine the allelic content of each sample on the plate. Mismatches between a probe and target reduce the efficiency of probe hybridization. Furthermore, AmpliTaq Gold DNA polymerase is more likely to displace the mismatched probe than to cleave it, releasing the reporter dye. By running the extension phase of the PCR at the optimal annealing temperature for the probes, the lower melting temperatures (Tm) for mismatched probes minimizes their cleavage and consequently their fluorescent contribution. The figure below illustrates results from matches and mismatches between target and probe sequences in TaqMan® PDARs for AD assays (Livak et al., 1995; Livak et al., 1999). Allele X F V Probe-target sequence match Match Allele Y Probe-target sequence mismatch Mismatch (higher Tm) V VIC F FAM Q Quencher V F Q Q Match Probe-target sequence match Legend Q Q Mismatch Probe-target sequence mismatch (higher Tm) AmpliTaq Gold DNA Polymerase GR1556 The table below shows the correlation between fluorescence signals and sequences present in the sample. A substantial increase in… 5-4 End-Point Analysis Indicates… VIC™ fluorescence only homozygosity for Allele X. FAM™ fluorescence only homozygosity for Allele Y. both fluorescent signals heterozygosity. Algorithmic Manipulation of Raw Allelic Discrimination Data The SDS software can analyze raw data immediately upon completion of an allelic discrimination run. The term ‘raw data’ refers to the spectral data between 500 nm to 660 nm collected by the SDS software during the plate-read. During the analysis, the software employs several mathematical algorithms to generate from the raw data a more direct measure of the relationship between the spectra changes in the unknown samples. The first mathematical algorithm involves the conversion of the raw data, expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye components using the extracted pure dye standards. After the dye components have been identified, the software determines the contribution of each dye in the raw data using the multicomponent algorithm. See “Multicomponenting” on page A-5 for a complete description of the process. Cluster Variations The SDS software graphs the results of an allelic discrimination run on a scatter plot contrasting reporter dye fluorescence (Allele X Rn versus Allele Y Rn). The software represents each well of the 384-well plate as a datapoint on the plot. The clustering of these datapoints can vary along the horizontal axis (Allele X), vertical axis (Allele Y), or diagonal (Allele X/Allele Y). This variation is due to differences in the extent of PCR amplification, which could result from differences in initial DNA concentration. The example below shows the variation in clustering due to differences in the extent of PCR amplification. Allele Y homozygotes Allele X/Allele Y heterozygotes Outliers Allele X homozygotes No amplification End-Point Analysis 5-5 Genotypic Segregation of Datapoint Clusters The figure on the previous page illustrates the concept of genotypic segregation of samples within the allele plot. The plot contains four separate, distinct clusters which represent the No Template Controls and the three possible genotypes (allele X homozygous, allele Y homozygous, and heterozygous). Because of their homogenous genetic compliment, homozygous samples exhibit increased fluorescence along one axis of the plot (depending on the allele they contain). In contrast, heterozygous samples appear within the center of the plot because they contain copies of both alleles, and therefore exhibit increased fluorescence for both reporter dyes. About Outliers Samples that did not cluster tightly may: 5-6 End-Point Analysis ♦ Contain rare sequence variations ♦ Contain sequence duplications ♦ Not contain a crucial reagent for amplification (the result of a pipetting error) Before You Begin Using SDS For specific instructions on any procedure described within this section, refer to the Online Help online help accompanying the SDS software. To get help at any time during the procedure, click a help button ( you are working. ) located within the dialog box or window in which Examples in The illustrations and screenshots that appear within this chapter were created from a This Section plate containing Pre-Developed TaqMan Assays and Reagents for Allelic Discrimination run to screen 6 human genomic DNA samples (HD 1, 2, 3, 4, 7, 8) for 2 targets (CYP 2C9*2 and CYP 2C19*2). Each well of the plate contains 1 µL DNA, 1X TaqMan® Universal PCR Master Mix, forward and reverse primers, and FAM and VIClabeled TaqMan probes. The following figure illustrates the arrangement of the assays, unknown samples, and no template control (NTC) wells on the plate. HD-1 1 HD-1 2 HD-1 3 HD-1 4 HD-1 5 HD-1 6 HD-1 7 HD-1 8 HD-1 9 HD-1 10 HD-2 1 HD-2 2 HD-2 3 HD-2 4 HD-2 5 HD-2 6 HD-2 7 HD-2 8 HD-2 9 HD-2 10 HD-3 1 HD-3 2 HD-3 3 HD-3 4 HD-3 5 HD-3 6 HD-3 7 HD-3 8 HD-3 9 HD-3 10 HD-4 1 HD-4 2 HD-4 3 HD-4 4 HD-4 5 HD-4 6 HD-4 7 HD-4 8 HD-4 9 HD-4 10 HD-7 1 HD-7 2 HD-7 3 HD-7 4 HD-7 5 HD-7 6 HD-7 7 HD-7 8 HD-7 9 HD-7 10 HD-8 1 HD-8 2 HD-8 3 HD-8 4 HD-8 5 HD-8 6 HD-8 7 HD-8 8 HD-9 9 HD-8 10 NTC GR2107 Not in Use PDAR CYP 2C9*2 PDAR CYP 2C19*2 Note The probes used in the example experiment were designed using the Primer Express™ Primer Design Software and by following the guidelines explained in “Assay Development Guidelines” on page C-2. IMPORTANT The SDS software does not require that allelic discrimination plates contain positive controls. End-Point Analysis 5-7 Analysis Checklist Where You Are in The following checklist illustrates your current position in the overall procedure: the Procedure Done Step Procedure See Page ✓ 1 Create an allelic discrimination plate document. 4-6 ✓ 2a a. Create detectors for the allelic discrimination probes. 4-7 b. Create a marker for each allelic discrimination probe pairing. 4-9 c. Copy the marker(s) to the plate document. 4-10 ✓ 3a Assign detector tasks to the wells of the plate document (NTC and Unknown). 4-11 ✓ 4 If you would like to perform thermal cycling of the allelic discrimination plate on the 7900HT instrument, create a real-time plate document for the plate and program it with the method for the allelic discrimination run. Otherwise, continue to step 5. 4-13 ✓ 5 Choose from the following: If running… Then… a single plate continue to step 7. the first plate in a series of plates with identical assay configurations Save the plate document as an ABI PRISM SDS Template Document as explained on page 4-17. ✓ 6 Create a plate document from the template created in step 5. 4-18 ✓ 7 Configure the document with sample names and plate information. 4-19 ✓ 8 a. Prepare the allelic discrimination plate or plates and perform thermal cycling on a designated thermal cycler. 4-20 b. Run the allelic discrimination plate or plates on the 7900HT instrument. 9 Analyze the run data. 5-9 10 View the results of the allelic discrimination run. 5-10 11 Call allele types for each marker. 5-11 12 Scrutinize the allele calls. 5-13 13 Choose from the following post-analysis options: 5-15 ♦ Reanalyze the run data. ♦ Adjust the display settings for the plate document. ♦ Print elements of the plate document. ♦ Export the plate document results table or plots. a. Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab-delimited text file. See “Importing Plate Document Setup Table Files” on page B-2 for more information. 5-8 End-Point Analysis Analyzing a Completed Allelic Discrimination Run Analyzing the Run To analyze a completed allelic discrimination run: Step Action 1 If not already open, launch the SDS software as explained on page 4-5. 2 Open the plate document file for analysis as follows: a. From the File menu, select Open. b. From the Look in text field, navigate to and select the plate document file. c. Click Open. The SDS software displays the plate document file. 3 Choose one of the following: ♦ From the Analysis menu, select Analyze. ♦ From the toolbar, click the Analyze button ( ). The SDS software analyzes the run data and displays the results in the Results tab. End-Point Analysis 5-9 Calling and Scrutinizing Allelic Discrimination Data About the Allelic The SDS software graphs the results of allelic discrimination runs on a scatter plot Discrimination View contrasting reporter dye fluorescence. After signal normalization and multicomponent analysis, the software graphs the normalized data from each well as a single data point on the plot. The following figure illustrates the components of the Allelic Discrimination plot. 1 2 3 4 The following table describes the elements of an SDS plate document: Component Description 1 Marker drop-down list Determines the marker data that the software displays within the plot. 2 Call drop-down list When a datapoint is selected, this menu allows you to assign an allele call to the datapoint within the scatter plot. 3 Toolbar Contains the following tools for manipulating the plot. Icon Description Selects individual data points by clicking or groups of datapoints by clicking and dragging a box across a group of data points. Selects groups of datapoints by encircling them with the tool. Repositions the view within the plot by clicking and dragging the screen. Zooms the plot by clicking the mouse button within the plot or by clicking and dragging a section of the plot to view. Zooms out on the plot by clicking the mouse button within the plot. 4 5-10 End-Point Analysis Scatter plot A scatter plot of data points from the run. Calling Allele Types To call allele types: Step 1 Action Click the Results tab. The software displays the Allelic Discrimination Plot. 2 Zoom out until all crossmarks are visible in the plot. a. Click the ( ) magnifying glass tool. b. Click the plot to zoom out. c. Click the ( ) lasso tool. d. Select all of the marks within the plot by clicking and dragging the mouse pointer across all datapoints in the plot. The software outlines all selected wells within the grid view. e. Examine the tray pane to confirm that all wells are selected. If not all wells are selected, repeat steps a-d until all wells are visible on the plot. Black boarder (surrounding selected wells) 3 Select the sample cluster exhibiting amplification of the first probe. Allele X homozygotes End-Point Analysis 5-11 To call allele types: Step 4 (continued) Action From the Call drop-down list, select the Allele X call. Select Allele X The software automatically labels the samples and wells with the Allele X call. Allele X homozygotes 5 Repeat steps 3 and 4 to apply calls to the rest of the samples within the plot. Call Symbol Definition Allele X ● Homozygous for the allele displayed on the X-axis of the Allelic Discrimination Plot. Allele Y ● Homozygous for the allele displayed on the Y-axis of the Allelic Discrimination Plot. Both ● Heterozygous (Alleles X and Y) NTC ■ No Template Control Undetermined ✕ Unknown (Unlabeled) Note You can adjust the appearance of the allelic discrimination plot or the data points it contains using the Display Settings dialog box. See the SDS software online help for more information. Allele Y homozygotes Allele X/Allele Y heterozygotes Allele X homozygotes No amplification 6 If evaluating for multiple markers, do the following: a. From the Marker drop-down list, select a different marker. b. Repeat steps 2 to 5 for the new marker. c. Repeat steps a and b until the alleles for each marker have been called. 5-12 End-Point Analysis Scrutinizing the To analyze allele types: Allele Calls Step 1 Action Verify the calls for the NTC, Allele X, and Allele Y controls. a. From the plate grid, select the wells containing the No Template Control samples. The software highlights the datapoints within the allele plot. b. Check that the datapoints cluster in the expected position on the plot. c. If using positive controls, repeat steps a and b for the wells containing the Allele X, and Allele Y controls. Allele Y controls should cluster here Allele X controls should cluster here No Template Controls should cluster here 2 Designate samples that did not cluster tightly as Undetermined. Samples that did not cluster tightly may: ♦ Contain rare sequence variations ♦ Contain sequence duplications Undetermined samples End-Point Analysis 5-13 To analyze allele types: Step 3 (continued) Action Screen for Unknown samples that failed to amplify: a. From the Allelic Discrimination Plot, select the NTC cluster. The SDS software highlights the datapoints within the allele plot and the plate grid. b. From the plate grid, check the wells containing Unknown samples for selected wells that are clustered with the NTCs. Samples that clustered with the No Template Control wells may: ♦ Contain no DNA ♦ Contain PCR inhibitors ♦ Be homozygous for a sequence deletion NTC cluster (selected) No Template Control wells Unknown samples clustered with the NTCs 4 Retest any samples that did not cluster tightly or clustered with NTCs to confirm the results. 5 If evaluating for multiple markers, do the following: a. From the Marker drop-down list, select a different marker. b. Repeat steps 1 to 4 for the new marker. c. Repeat steps a and b until the calls for each marker have been verified. 5-14 End-Point Analysis After the Analysis Changing the Before printing or exporting the analyzed data, the software allows you to reconfigure Plate Document the appearance of several elements of the plate document including the results table, Display Settings plate grid, and most plots (Allelic Discrimination, Raw Data, and Background plots). To configure the display settings for the plate document: Step Action 1 From the View menu, select Display Settings. 2 From the Display Settings dialog box, click the help button ( instructions on modifying the display settings. ) for further Saving the The software allows you to save any changes to the appearance of the plate Plate Document document, however it does not save the calls made during the analysis. To save the plate document, select Save from the File menu. Printing a Report The SDS software can print a report of the analyzed data containing individual or multiple elements of the plate document. To print a report of the plate document data: Step Action 1 From the File menu, select Print Report. 2 From the Print Report dialog box, click the help button ( setting up, previewing, and printing the report. ) for instructions on Exporting Plate Exporting Plate Document Data as a Tab-Delimited Text File Document Data The SDS software can export raw or analyzed data in tab-delimited (*.txt) format for all or a select group of wells on a plate document. The exported files are compatible with most spreadsheet applications and programs that can read tab-delimited text. To export run data as a tab-delimited text file, choose one of the following for further instructions: ♦ See “Exporting Plate Document Data” on page B-9. ♦ Click the help button ( ) within the table view. Help button Exporting Plots as Graphics The SDS software can export most panes and plots of the plate document as JPEG (Joint Photographic Experts Group) graphic files. The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software. To export a plot as a graphic file, see “Exporting Graphics” on page B-8 or click the help button ( ) within the plot of interest for further instructions. End-Point Analysis 5-15 Real-Time Analysis 6 6 In This Chapter This chapter discusses the following topics: Topic Real-Time Runs on the 7900HT Instrument See Page 6-2 Section: Absolute Quantification 6-3 Overview 6-4 Before You Begin 6-6 Analysis Checklist 6-7 Analyzing the Run Data 6-8 Viewing Results 6-13 After the Analysis 6-15 Section: Dissociation Curve Analysis 6-17 Overview 6-18 Before You Begin 6-19 Analysis Checklist 6-20 Analyzing the Run Data 6-21 Determining Tm Values for the Analyzed Run 6-22 After the Analysis 6-24 Real-Time Analysis 6-1 Real-Time Runs on the 7900HT Instrument Real-Time Runs Real-time is the term used to describe the category of sequence detection runs in which the ABI PRISM® 7900HT Sequence Detection System is used to measure the fluorescence of a biological sample during thermal cycling. In contrast to end-point runs, real-time experiments can be used to achieve both qualitative and quantitative measurements. Real-time analysis can be used in combination with either TaqMan® or SYBR® Green 1 double-stranded DNA binding dye chemistry for a variety of purposes including quantitative PCR and dissociation curve analysis. Quantitative Quantitative RT-PCR is a method used to measure small quantities of ribonucleic acid RT-PCR sequences isolated from biological samples. Typical biological samples include cells, tissues, and fluids. During the RT step, reverse transcription of target RNA produces corresponding complementary DNA (cDNA) sequences. During the subsequent PCR, the initial concentration of target cDNA is quantified by amplifying it to a detectable level. There are two types of quantitative RT-PCR: 6-2 Real-Time Analysis ♦ Absolute quantification ♦ Relative quantification Section: Absolute Quantification In This Section This section contains the following information: Topic See Page Overview 6-4 Before You Begin 6-6 Analysis Checklist 6-7 Analyzing the Run Data 6-8 Viewing Results 6-13 After the Analysis 6-15 Real-Time Analysis 6-3 Overview About Absolute The ABI PRISM 7900HT Sequence Detection System supports real-time absolute Quantification quantification of nucleic acids using a standard curve method. The objective of absolute quantification is to accurately determine the absolute quantity of a single nucleic acid target sequence within an unknown sample. The results of an absolute quantification experiment are reported in the same unit measure of the standard used to make them. Employing the Absolute quantification on the 7900HT instrument is accomplished through the use of 5´ Nuclease Assay the polymerase chain reaction and the fluorogenic 5´ nuclease assay (see page A-2). During setup, standards diluted over several orders of magnitude and unknown samples are loaded onto an ABI PRISM® Optical Reaction Plate containing master mix and TaqMan assays targeting a specific nucleic acid sequence. The plate is then loaded into a 7900HT instrument which has been configured to perform a real-time run. During the thermal cycling, the instrument records the emission resulting from the cleavage of TaqMan® probes in the presence of the target sequence. After the run, the SDS software processes the raw fluorescence data to produce threshold cycle (CT) values for each sample (see page A-10). The software then computes a standard curve from the CT values of the diluted standards and extrapolates absolute quantities for the unknown samples based on their CT values (see below). Note See Appendix A, “Theory of Operation,” for more information on the fluorogenic 5´ nuclease assay, real-time data collection, or the mathematical transformations of sequence detection data. Standard Curve Plot 28 26 CT Unknowns 24 22 20 1.0 E+2 1.0 E+3 1.0 E+4 1.0 E+5 Quantity (LogN initial concentration) The figure above illustrates a standard curve generated from a standard RNase P Installation Plate. The arrangement of the samples and standards on the plate are shown in “Examples in This Chapter” on page 6-6. 6-4 Real-Time Analysis Algorithmic The SDS software can analyze raw data immediately upon completion of absolute Manipulation of quantification run. The term raw data refers to the spectral data between 500 nm to Raw Data 660 nm collected by the Automation Controller Software during the plate-read. During the analysis, the software automatically applies several mathematical transformations to the raw data to generate a more direct measure of the relationship between the spectral changes in the unknown samples. Multicomponenting The first mathematical transformation involves the conversion of the raw data, expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye components using the extracted pure dye standards. After the dye components have been identified, the software determines the contribution of each dye in the raw data using the multicomponent algorithm. See “Multicomponenting” on page A-5 for a complete description. Setting the Threshold and Calling CTs After multicomponenting, the baseline and threshold values must be set for the run (see “Kinetic Analysis/ Quantitative PCR” on page A-7 for more information). The results of the experiment can be visualized in the Standard Curve graph of the Results tab. The graph consists of a scatter plot of standard and unknown samples graphed on a linear-scale plot of Threshold Cycle (CT) versus Starting copy number. Real-Time Analysis 6-5 Before You Begin Using SDS For specific instructions on any procedure described within this section, refer to the Online Help online help accompanying the SDS software. To get help at any time during the procedure, click a help button ( you are working. ) located within the dialog box or window in which Examples in This The illustrations and screenshots that appear within this chapter were created for a Chapter TaqMan® RNase P Instrument Verification Plate, an experiment run during the installation of the 7900HT instrument to verify its performance. The sealed plate is pre-loaded with the reagents necessary for the detection and quantification of genomic copies of the human RNase P gene (a single-copy gene encoding the moiety of the RNase P enzyme). Each well contains pre-loaded reaction mix (1X TaqMan® Universal PCR Master Mix, RNase P primers, and FAM™-labeled probe) and template. Unknown 2 10000 GR2107 Unknown 1 5000 NTC STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 The following figure illustrates the arrangement of standards and samples on the RNase P plate. As shown below, the RNase P plate consists of 5 columns of template standards (1250, 2500, 5000, 10,000, and 20,000 copies) and two unknown populations (5000 and 10,000 copies). 6-6 Real-Time Analysis Analysis Checklist Where You Are in The following checklist illustrates your current position in the overall procedure: the Procedure Done Step ✓ 1 Create an absolute quantification plate document. 4-6 ✓ 2a a. Create detectors for the absolute quantification probes. 4-7 ✓ 3a ✓ ✓ 4 5 Procedure See Page b. Copy the detectors to the plate document. 4-8 a. Configure the plate document with detector tasks (NTC, Standard, and Unknown). 4-11 b. Assign quantities to the wells of the plate document that contain standards. 4-12 a. Program the method for the absolute quantification run. 4-13 b. If performing an assay in which you would like to collect dissociation data, add a temperature ramp to the thermal profile to perform a dissociation curve analysis. 4-16 Choose from the following: If running… Then… a single plate continue to step 7. the first plate in a series of plates with identical assay configurations Save the plate document as an ABI PRISM SDS Template Document as explained on page 4-17. ✓ 6 Create a plate document from the template created in step 5. 4-18 ✓ 7 Configure the document with sample names and plate information. 4-19 ✓ 8 Prepare and run the absolute quantification plate or plates. 4-20 9 Configure the analysis options for the run. 6-8 10 Analyze the run data. 6-9 11 Set the baseline and threshold values for each detector. 6-10 12 Visualize outliers and eliminate any outlying amplification from the run data. 6-12 13 View the results of the absolute quantification run. 6-13 14 Choose from the following post-analysis options: 6-15 ♦ Reanalyze the run data. ♦ Adjust the display settings for the results table, plate grid, and plate document plots. ♦ Print elements of the plate document. ♦ Export the plate document results table or plots. a. Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab-delimited text file. See “Importing Plate Document Setup Table Files” on page B-2 for more information. Real-Time Analysis 6-7 Analyzing the Run Data Configuring the Before analyzing the data collected from the complete absolute quantification run, Analysis Options decide whether to configure the options for the software analysis. The analysis (Optional) options allow you to pre-configure the threshold and baseline settings applied to the plate document data during the analysis. If you choose not to configure the analysis options, the SDS software uses the default baseline range and automatically calculates the threshold value for the run. To configure the analysis options for the absolute quantification run: Step Action 1 If not already open, launch the SDS software as explained on page 4-5. 2 Open the plate document file for analysis as follows: a. From the File menu, select Open. b. From the File Type drop-down list, select ABI PRISM SDS Single Plate (*.sds). c. From the Look in text field, navigate to and select the plate document file. d. Click Open. The SDS software displays the plate document file. 3 From the Analysis menu, select Analysis Options. The Analysis Options - Absolute Quantification dialog box appears. 4 If desired, click the Threshold text field, and type an initial threshold value to use for the analysis of the plate document. For more information about the threshold setting, see “Calculating Threshold Cycles” on page A-10. Note If no threshold is specified, the SDS software will automatically assign a threshold value during the analysis. 5 If desired, set a default baseline for the analysis: a. Click the Baseline Start text field, and type or dial an initial baseline to use for the analysis of the plate document. b. Click the Stop text field, and type or dial an stop baseline to use for the analysis of the plate document. Note If no baseline is specified, the SDS software will use the default baseline range of cycles 3-15 for the analysis. 6 Set the baseline and threshold values for any remaining detectors present on the plate as follows: a. From the Detector drop-down list, select another detector. b. Repeat steps 4 and 5 until the baseline and threshold values have been set for each detector. 7 Click OK. The software closes the dialog box and configures the analysis with new settings. 6-8 Real-Time Analysis Analyzing the Run Once you have configured the analysis options, you can analyze the run data. During the analysis, the software mathematically transforms the raw data to establish a comparative relationship between the spectral changes in the passive reference dye and those of the reporter dye. Based on that comparison, the software calculates a cycle threshold (CT) for each reaction (standard and unknown). The software then generates a standard curve for the run by plotting the standard samples on a graph of CT versus initial copy number. Note See Appendix A, “Theory of Operation,” for a detailed description of the SDS software mathematical transformation of real-time run data. To analyze the run: Step 1 Action Select all wells in the plate grid. The software outlines the selected wells with a black line. 2 Choose one of the following: ♦ From the Analysis menu, select Analyze. ♦ From the toolbar, click the Analyze button ( ). The SDS software analyzes the run data and displays the results in the Results tab. 3 Set the baseline and threshold values for each detector on the plate as explained on page 6-10. Real-Time Analysis 6-9 Setting the Baseline Before calculating absolute quantification values, the baseline and threshold values and Threshold must be set for all detectors present on the plate. Values for the Run To set the baseline and threshold values for the run: Step 1 Action Click the Results tab. The software displays the contents of the Results tabbed page. 2 If the Amplification Plot is not visible, click the Amplification Plot button ( ). The software displays the results of the sequence detection run in an amplification plot of normalized reporter fluorescence (Rn) versus threshold cycle (CT). 3 From the Plot drop-down list, select Rn vs. Cycle. The software plots the analyzed data in the graph of reporter fluorescence versus cycle number. 4 Identify the components of the linear scale amplification plot and set the baseline so that the amplification curve growth begins at a cycle number greater than the Stop baseline cycle. IMPORTANT Do not adjust the default baseline if the amplification curve growth begins after cycle 15. If the amplification plot looks like... Then... the amplification curve begins after the maximum baseline (cycle 15). Do not adjust the baseline. the maximum baseline is set too high. Decrease the Stop baseline value by clicking and dragging the right carrot to an earlier cycle. the maximum baseline is set too low. Increase the Stop baseline value by clicking and dragging the right carrot to a later cycle. 6-10 Real-Time Analysis To set the baseline and threshold values for the run: Step 5 (continued) Action From the Plot drop-down list, select ∆Rn vs. Cycle. The software plots the analyzed data in the graph of normalized reporter fluorescence (∆Rn) versus cycle number. 6 Identify the components of the amplification curve and set the threshold so that it is: ♦ Above the background ♦ Below the plateaued and linear regions of the amplification curve ♦ Within in the geometric phase of the amplification curve Plateau phase Linear phase Geometric phase Threshold setting (click and drag) Rn Background Cycle 7 Baseline Set the baseline and threshold for any remaining detectors present on the plate as follows: a. From the Detector drop-down list, and select another detector. b. Repeat steps 3 to 6 until the baseline and threshold values have been set for each detector. 8 Eliminate outliers from the analyzed run data as explained on page 6-12. Real-Time Analysis 6-11 Eliminating Outliers For any PCR, experimental error may cause some wells to amplify insufficiently or not at all. These wells typically produce CT values that differ significantly from the average for the associated replicate wells. If included in the absolute quantification calculations, these outliers can potentially result in erroneous measurements. Visualizing Outliers To ensure precise absolute quantification, replicate groups must be carefully scrutinized for outlying wells. The CT vs. Well Position view of the Amplification Plot allows you to examine each set of replicate wells for outliers. To visualize the replicate groups for outlying amplification: Step 1 Action From the Plot drop-down list, select Ct vs. Well Position. The SDS software displays the results data in a Well versus Threshold Cycle plot. 2 Verify the uniformity of each replicate population by comparing the groupings of CT values for the wells that comprise the set. Are outliers present? Then... Yes a. Determine and record the well numbers of all outlying wells. b. Go to the next step. No 3 go to the next step. Check for remaining detectors present on the plate for outliers: a. Click the Detector drop-down list, and select another detector. b. Repeat step 2 until each detector has been checked for outliers. 4 If outliers are present in your data, eliminate them as explained below. Otherwise, go on to “Viewing Results” on page 6-13. Eliminating Wells from the Analysis IMPORTANT If one or more wells are removed from use before a plate document has been run, the SDS software will not collect data for those wells. If you identified any outliers in the previous procedure, you must eliminate them from the standard curve calculation before viewing the results. To eliminate outliers from the calculations: Step 1 Action While pressing and holding the Ctrl key, click each well in the plate grid that you identified as an outlier in the previous procedure. The SDS software creates a black boarder around each cell as it is selected. 6-12 Real-Time Analysis 2 Click the Setup tab. 3 From the well inspector of the Setup tabbed page, uncheck the In Use check box. 4 From the Analysis menu, select Analyze. 5 Reapply the baseline and threshold values determined on page 6-10. Viewing Results Viewing the Displays the results of the absolute quantification calculation in the results table of the Analysis Table plate document. The figure below shows an example of the results table containing the data from a TaqMan RNase P Instrument Verification Plate. Help button Table elements: Column Displays Position The coordinate position of the well on the plate. Sample Name The sample name applied to the well. Note See page 4-19 for information on applying sample names to the plate document. Detector The name of the detector assigned to the well. Note See page 4-7 for information on applying detectors to the plate document. Task The task (NTC, Standard, or Unknown) assigned to the well. Note See page 4-11 for information on applying detector tasks to the plate document. CT The threshold cycle generated by the well during the PCR. Quantity ♦ For wells containing Unknown samples, this column displays the starting copy number for the well calculated by the software from the standard curve. ♦ For wells containing Standard, this column displays the quantity assigned to the well. Note See page 4-11 for information on applying quantities to standard wells of the plate document. The following two columns contain data only if a well is run as part of a replicate group. Qty Mean The arithmetic mean for the quantity values of the replicate group associated with the well. Qty stddev The standard deviation of the quantity values of the replicate group associated with the well. Real-Time Analysis 6-13 Viewing the The software displays the standard curve generated from the run data within the Standard Curve Results tab of the plate document. The standard curve plot displays the unknown samples on a graph of CT (threshold cycle) vs. initial quantity (LogN). The following figure illustrates the components of the standard curve plot. Help button Detector drop-down list Legend box Hide Unknowns button Standard Curve box Standard curve plot The following table describes the elements of an SDS plate document: Component Description Help button Launches the SDS software online help. Detector menu Toggles the data displayed within the plot based on detector name. Legend box Displays a symbol key for the datapoints appearing in the plot. Hide Unknowns button Toggles the presence of data from unknown samples in the plot. Standard Curve box Contains statistical data describing the standard curve. Item Definition Slope The slope of the standard curve. The slope of the standard curve is useful for assessing the efficiency of the assay. At 100% efficiency, a reaction should achieve a slope of −3.33 since every 10-fold difference in quantity translates to a difference of 3.33 CTs. Y Inter The Y-axis intercept of the standard curve. R2 The R Square value for the standard curve that describes the correlation between threshold cycles (CT) and the log of the starting copy number for the samples that comprise the standard curve plot. The calculation yields a value between 1 and 0, where values closer to 1 indicate better correlation between CT and the log of the starting copy number. Note The software calculates the R Square value by taking the square of the Pearson Coefficient of Correlation (also known as the r value) calculated for the data points that comprise the plot. The software calculates the R2 value only for the standards that make up the curve. Standard curve plot 6-14 Real-Time Analysis A scatterplot of datapoints from the absolute quantification run. After the Analysis Changing the Before printing or exporting the analyzed data, the software allows you to reconfigure Plate Document the appearance of several elements of the plate document including the results table, Display Settings plate grid, and most plots. To configure the display settings for the plate document: Step Action 1 From the View menu, select Display Settings. 2 From the Display Settings dialog box, click the help button ( instructions on modifying the display settings. ) for further Saving the The software allows you to save any changes made the display settings and/or Plate Document analysis settings (baseline and threshold values) of the plate document. To save the analyzed plate document, select Save from the File menu. IMPORTANT The Save command saves does not save the results of the analysis, only the analysis settings. Saved plate documents must be re-analyzed each time they are opened. Printing a Report The SDS software can print a report of the analyzed data containing individual or multiple elements of the plate document. To print a report of the plate document data: Step Action 1 From the File menu, select Print Report. 2 From the Print Report dialog box, click the help button ( setting up, previewing, and printing the report. ) for instructions on Exporting Plate Exporting Plate Document Data as a Tab-Delimited Text File Document Data The SDS software can export raw or analyzed data in tab-delimited (*.txt) format for all or a select group of wells on a plate document. The exported files are compatible with most spreadsheet applications and programs that can read tab-delimited text. To export run data as a tab-delimited text file, choose one of the following for further instructions: ♦ See “Exporting Plate Document Data” on page B-9. ♦ Click the help button ( ) within the table view. Exporting Plots as Graphics The SDS software can export most panes and plots of the plate document as JPEG (Joint Photographic Experts Group) graphic files. The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software. To export a plot as a graphic file, see “Exporting Graphics” on page B-8 or click the help button ( ) within the plot of interest for further instructions. Real-Time Analysis 6-15 6-16 Real-Time Analysis Section: Dissociation Curve Analysis In This Section This section contains the following information: Topic See Page Overview 6-18 Before You Begin 6-19 Analysis Checklist 6-20 Analyzing the Run Data 6-21 Determining Tm Values for the Analyzed Run 6-22 After the Analysis 6-24 Real-Time Analysis 6-17 Overview About Dissociation The ABI PRISM 7900HT Sequence Detection System supports dissociation curve Curve Analysis analysis of nucleic acids using SYBR® Green 1 double-stranded DNA binding dye chemistry. The objective of dissociation curve analysis is to accurately determine the melting temperature (Tm) of a single target nucleic acid sequence within an unknown PCR sample. Typical uses of dissociation curves include detection of non-specific products and primer concentration optimization. Employing the Dissociation curve analysis on the 7900HT instrument is made possible through the SYBR Green 1 Dye use of the fluorogenic SYBR Green 1 double-stranded DNA binding dye chemistry (see page A-3). Dissociation curves are commonly performed following the PCR stage of a SYBR Green experiment to screen for non-specific products. To generate the data needed to create a curve, the 7900HT instrument performs a programmed temperature ‘ramp’ in which it slowly elevates the temperature of the plate over several minutes. The specific binding characteristic of the SYBR Green 1 Dye permits the 7900HT instrument to monitor the hybridization activity of the nucleic acids present in the sample. During the run, the instrument records the decrease in SYBR Green fluorescence resulting from the dissociation of dsDNA. Mathematical After the run, the SDS software processes the raw fluorescence data from the SYBR Transformations Green 1 Dye to generate a more meaningful representation of the relationship between spectral change and temperature for the dissociation curve run. Multicomponenting and Normalization The first mathematical transformation involves the conversion of the raw data, expressed in terms of Fluorescent Signal vs. Wavelength, using the extracted pure dye standards, to pure dye components. After the dye components have been identified, the software determines the contribution of each dye in the normalized data using the multicomponent algorithm (see “Multicomponenting” on page A-5 for a complete description of the process). Finally, the software normalizes the data using the component of the passive reference dye as shown below. R ( SYBR ) R n = ---------------------------------------------R ( PassiveReference ) Derivation of Dissociation Curve Data The SDS software then computes the first derivative of the normalized data (Rn) for each reading taken by the 7900HT instrument during the temperature ramp. The resulting derivative data (Rn´) is the rate of change in fluorescence as a function of temperature (see below). dR n R n ′ = --------dT The software plots the negative of the resulting derivative data on graph of -Rn´ versus temperature (T) that visualizes the change in fluorescence at each temperature interval. The Tm for the target nucleic acid can be determined from the graph by identifying the maximum for the rate of change (displayed as a peak) for the appropriate amplification curve. 6-18 Real-Time Analysis Example Results The following figure illustrates a typical dissociation curve from an experiment run to detect non-specific amplification in cDNA samples. -6.0 Primer Dimer Tm = 74.9 °C -5.0 Main Product Tm = 80.5 °C -Rn -4.0 -3.0 -2.0 -1.0 0.0 60 65 70 75 80 Temperature (°C) 85 90 95 The plot above displays the dual amplification peaks typical of primer-dimer formation. The amplification from the specific product is displayed with a Tm of 80.5 °C, while the primer-dimer product has a characteristically lower Tm of 74.9 °C. Before You Begin Using SDS For specific instructions on any procedure described within this section, refer to the Online Help online help accompanying the SDS software. To get help at any time during the procedure, click a help button ( you are working. ) located within the dialog box or window in which Examples in This The illustrations and screenshots that appear within this chapter were created from a Chapter plate run to determine the purity of a β-actin amplification in unknown samples. Each well of the plate contains SYBR Green 1 dye, forward and reverse primers, and genomic DNA known to contain complimentary binding sites. Real-Time Analysis 6-19 Analysis Checklist Where You Are in The following checklist illustrates your current position in the overall procedure: the Procedure Done Step ✓ 1 Create an absolute quantification plate document. 4-6 ✓ 2a a. Create detectors for the absolute quantification probes. 4-7 ✓ 3a ✓ ✓ 4 5 Procedure See Page b. Copy the detectors to the plate document. 4-8 a. Configure the plate document with detector tasks (NTC, Standard, and Unknown). 4-11 b. Assign quantities to the wells of the plate document that contain standards. 4-12 a. Program the method for the absolute quantification run. 4-13 b. Add a temperature ramp to the thermal profile. 4-16 Choose from the following: If running… Then… a single plate continue to step 7. the first plate in a series of plates with identical assay configurations Save the plate document as an ABI PRISM SDS Template Document as explained on page 4-17. ✓ 6 Create a plate document from the template created in step 5. 4-18 ✓ 7 Configure the document with sample names and plate information. 4-19 ✓ 8 Prepare and run the dissociation curve plate or plates. 4-20 9 Analyze the run data. 6-21 10 View the results of the dissociation curve analysis. 6-22 11 Determine melting temperature (Tm) values for the derivative peaks. 6-23 12 Choose from the following post-analysis options: 6-24 ♦ Reanalyze the run data. ♦ Adjust the display settings for the results table, plate grid, and plate document plots. ♦ Print elements of the plate document. ♦ Export the plate document results table or plots. a. Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab-delimited text file. See “Importing Plate Document Setup Table Files” on page B-2 for more information. 6-20 Real-Time Analysis Analyzing the Run Data Analyzing the Run The run data from a temperature ramp can be analyzed immediately following the completion of the run. For an explanation of how the software manipulates the raw data, see “Algorithmic Manipulation of Raw Data” on page 6-5. To analyze the run: Step Action 1 If not already open, launch the SDS software as explained on page 4-5. 2 Open the plate document file for analysis as follows: a. From the File menu, select Open. b. From the File Type drop-down list, select ABI PRISM SDS Single Plate (*.sds). c. From the Look in text field, navigate to and select the plate document file. d. Click Open. The SDS software displays the plate document file. 3 Select all wells in the plate grid. The software outlines the selected wells with a black line. 4 Choose one of the following: ♦ From the Analysis menu, select Analyze. ♦ From the toolbar, click the Analyze button ( ). The software analyzes the run data and displays the results in the Dissociation Curve tab. 5 Determine the Tm for the dissociation curves displayed within the Dissociation Curve tabbed page as explained on page 6-23. Real-Time Analysis 6-21 Determining Tm Values for the Analyzed Run Viewing Analyzed The SDS software displays the results of the dissociation curve analysis within the Dissociation Curve Dissociation Curve tab of the plate document. The tab displays the analyzed data in a Data graph of the negative of the derivative (-Rn´) versus temperature (T) that visualizes the change in fluorescence at each temperature interval during the ramp. Note The plot displays data from the selected wells of the plate grid. If you do not see dissociation curve data, select the wells of the plate grid containing the SYBR Green reactions. The following figure illustrates the components of the Dissociation Plot. Dissociation Curve tab Dissociation Plot Dissociation curves Tm display and slider Step drop-down list Plot drop-down list Detector drop-down list The following table describes the elements of the Dissociation Plot: Component Description Dissociation Plot The plot displays data from the selected wells in the plate grid. Note The properties of the Dissociation Plot are adjustable. For more information on adjusting the appearance of the plot, click the help button ( ) and see the SDS software online help. Step drop-down list Chooses the data displayed within the plot based on the ramp. If a plate document contains data from more than one temperature ramp, the Step drop-down list allows you to displays the data from each by selecting the position of the ramp in the thermal profile. Tm display and slider The SDS software displays the Tm below the green slider (see above). There are two definitions for the Tm value: ♦ The chemical definition is the temperature at which 50% of the DNA is in a double-stranded configuration. Tm 6-22 Real-Time Analysis ♦ The mathematical definition is the maximum value for the first derivative curve within a specific peak. The following table describes the elements of the Dissociation Plot: (continued) Component Description Plot drop-down list Chooses the data displayed within the plot based on the derivative calculation. The list offers the following selections: ♦ Raw – When selected, this option plots the normalized reporter fluorescence data (Rn) on a graph of fluorescence vs. temperature (see below left). ♦ Derivative – When selected, this option plots derivative data (Rn´) on a graph of the derivative vs. temperature (see below right). The derivative data is the negative of the rate of change in fluorescence as a function of temperature. The following figures show the plots accessible from the Plot drop-down list. Raw Plot Derivative Plot Chooses the data displayed within the plot based on detector name. Detector drop-down list Determining To determine the Tm value of a melting curve within the Dissociation Plot: Tm Values Step 1 Action Move the mouse pointer over the green Tm line located on the Y-axis line of the plot. The mouse pointer becomes a hand. 2 Click and drag the Tm line to the maximum point of the derivative plot of interest. The SDS software displays the Tm for the curve below the Tm line. Tm display and slider Note The apex of the curvature of represents the maximum rate of change in normalized fluorescence. Real-Time Analysis 6-23 After the Analysis Changing the Before printing or exporting the analyzed data, the software allows you to reconfigure Plate Document the appearance of several elements of the plate document including the results table, Display Settings plate grid, and most plots. To configure the display settings for the plate document: Step Action 1 From the View menu, select Display Settings. 2 From the Display Settings dialog box, click the help button ( instructions on modifying the display settings. ) for further Saving the The software allows you to save any changes to the appearance of the plate Plate Document document, however it does not save the threshold or baseline values made during the analysis. To save the plate document, select Save from the File menu. Printing a Report The SDS software can print a report of the analyzed data containing individual or multiple elements of the plate document. To print a report of the plate document data: Step Action 1 From the File menu, select Print Report. 2 From the Print Report dialog box, click the help button ( setting up, previewing, and printing the report. ) for instructions on Exporting Plate Exporting Plate Document Data as a Tab-Delimited Text File Document Data The SDS software can export raw or analyzed data in tab-delimited (*.txt) format for all or a select group of wells on a plate document. The exported files are compatible with most spreadsheet applications and programs that can read tab-delimited text. To export run data as a tab-delimited text file, choose one of the following for further instructions: ♦ See “Exporting Plate Document Data” on page B-9. ♦ Click the help button ( ) within the table view. Help button Exporting Plots as Graphics The SDS software can export most panes and plots of the plate document as JPEG (Joint Photographic Experts Group) graphic files. The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software. To export a plot as a graphic file, see “Exporting Graphics” on page B-8 or click the help button ( ) within the plot of interest for further instructions. 6-24 Real-Time Analysis System Maintenance 7 7 In This Chapter This chapter discusses the following topics: Topic See Page Recommended Maintenance Schedule 7-2 Section: Maintaining the 7900HT Instrument 7-3 Replacing the Sample Block 7-4 Changing the 7900HT Plate Adapter 7-9 Decontaminating the Sample Block 7-11 Performing a Background Run 7-13 Performing a Pure Dye Run 7-17 Adding Custom Dyes to the Pure Dye Set 7-21 Verifying Instrument Performance Using a TaqMan RNase P Plate 7-24 Section: Maintaining the Plate Handler 7-27 Adjusting the Sensitivity of the Plate Sensor Switch 7-28 Aligning the Plate Handler 7-32 Aligning the Fixed-Position Bar Code Reader 7-40 Cleaning and Replacing Gripper Finger Pads 7-43 Section: Maintaining the Computer and SDS Software 7-45 General Computer Maintenance 7-46 Maintaining the SDS software 7-48 System Maintenance 7-1 Recommended Maintenance Schedule Maintenance The following table includes a list of tasks that should be performed on the Schedule ABI PRISM® 7900HT Sequence Detection System regularly. Interval Task Weekly Archive SDS Files 7-46 Perform a Background Run 7-13 Check (and if necessary replace) Gripper Finger Pads 7-43 Defragment the Computer Hard Drive 7-47 Monthly Semi-Annually Perform a Pure Dye See Page Runa Check Applied Biosystems Web Site for Software Updates a. Perform a background run prior to each Pure Dye Run. 7-2 System Maintenance 7-17 7-48 Section: Maintaining the 7900HT Instrument In This Section This section contains the following information: Topic See Page Replacing the Sample Block 7-4 Changing the 7900HT Plate Adapter 7-9 Decontaminating the Sample Block 7-11 Performing a Background Run 7-13 Performing a Pure Dye Run 7-17 Adding Custom Dyes to the Pure Dye Set 7-21 Verifying Instrument Performance Using a TaqMan RNase P Plate 7-24 System Maintenance 7-3 Replacing the Sample Block When to Perform IMPORTANT Before changing the sample block, perform all required upgrades to the SDS software and instrument firmware. Failure to update the software can render the instrument inoperable or result in damage to instrument components. You will need to remove the 7900HT instrument sample block, when: ♦ Decontaminating the wells of the sample block (see page 7-11) ♦ Changing sample block formats IMPORTANT Always run a background plate after installing the sample block. Sample Block Unless instructed to do otherwise, adhere to the following guidelines when Installation exchanging sample block modules of different formats: Checklist To install a sample block module: Step Procedure See Page 1 Perform all required software and firmware upgrades to the ABI PRISM 7900HT Sequence Detection System. 7-48 2 Remove the existing sample block. 7-5 3 Install the new sample block. 7-7 4 Change the plate adapter. 7-9 5 Run a background plate to check the sample block for contamination. 7-13 If changing block formatsa or if installing a new block, also perform the following… 6 Run a pure dye plate to create the pure spectra calibration values for the new format. 7-17 7 Run an TaqMan® RNase P Instrument Verification Plate to confirm the proper operation of the sample block. 7-24 If using an automation accessory, also perform the following… 8 If changing consumable formatsa, adjust the plate sensor switch on the plate handler arm for the new plates. 7-28 9 Align the Zymark® Twister™ Microplate Handler fixed-position bar code reader for the new plate format. 7-32 Note It is necessary to align the plate handler to only the Instrument position (Zymark position 2) as explained on pages 7-32 to 7-35. 10 Align the fixed-position bar code reader for the new plate format. 7-40 a. For example, when replacing a 384-well sample block with a 96-well block Materials Required The procedure below requires the use of the following materials: Material 7-4 System Maintenance Part Number Replacement Sample Block (if replacing the sample block) — 5/32 inch Hex key (necessary only for certain instruments) — 5/16 inch Hex key (some instruments may require a crescent wrench) — Handling the The interchangeable sample blocks are delicate pieces of equipment containing Sample Block several fragile components that can break if handled improperly. The figure below illustrates the correct locations for handling the interchangeable sample block module. Circuitry and connections to the instrument (Do Not Touch) GR2028 Heat sinks Hold sample block module from the sides (Bottom of module) Removing the To remove the sample block: Sample Block Step 1 Action Launch the Automation Controller Software and perform the following tasks: a. Click the Thermal Status tab, and confirm the function of the current module. The module is operating normally if the software is receiving a temperature reading. b. Click Open/Close to rotate the instrument tray to the OUT position. 2 From the File menu, select Exit to close the Automation Controller Software. 3 Turn off and unplug the 7900HT instrument. ! WARNING PHYSICAL HAZARD. The instrument must be unplugged and turned off at all times during the following procedure. Failure to comply can result in serious physical injury to the user or damage to the instrument. 4 Wait 20–30 min for the heated cover to cool. ! CAUTION PHYSICAL HAZARD. During instrument operation, the temperature of the sample block can be as high as 100 °C. Before performing this procedure, wait until the sample block reaches room temperature. If the instrument tray is in the OUT position (outside of the instrument), push it into the instrument to provide an open work space. 6 If using a Zymark Twister Microplate Handler remove the covers for the fixed-position bar code reader and the underlying platform. GR2009 5 Push the instrument tray within the instrument, and remove the thermal cycler access cover to permit access to the sample block. Note The thermal cycler access cover is secured to the instrument by non-locking pins and may require force to remove it (no tools are required). GR2023b 7 Fixed-position bar code reader and underlying platform covers System Maintenance 7-5 To remove the sample block: Step 8 (continued) Action Using a 5/16 inch Hex key, turn the sample block locking bolt counter-clockwise until it is very loose, but still attached to the sample block locking bar. GR2024 Note Some instruments may require the use of an adjustable crescent wrench to loosen the sample block locking bolt. Sample block locking bar Sample block locking bolt GR2025b Loosen the thumb screw securing the sample block locking bar to the instrument chassis (may be a 5/32 Hex bolt on some instruments). GR2024b 9 Thumb screw Lift the sample block locking bar up and out of the instrument. 11 Remove the sample block from the instrument as follows: GR2025b 10 GR202 a. Rotate the release lever at the base of the sample block 90 degrees. GR2027 b. Being careful of the cooling fins on the bottom of the sample block, slide the sample block out of the instrument and place it on a clean, level surface. 7-6 System Maintenance Replacing the IMPORTANT Before changing the sample block, perform all required upgrades to the SDS Sample Block software and instrument firmware. Failure to upgrade the software can render the instrument inoperable or result in damage to instrument components. To replace the sample block: Step 1 Action Load the sample block into the instrument compartment as follows: a. Being careful of the heat sinks on the bottom of the sample block, rest the sample block on the metal runners on either side of the instrument bay. GR2027 b. Carefully slide the sample block into the instrument until the front of the block is flush with the rear of the locking bar. c. Once seated, firmly press on the sample block to ensure a good connection. Replace the sample block locking bar. 3 Tighten the thumb screw (from step 9 on page 7-6) to secure the sample block locking bar to the instrument chassis (may be a 5/32 Hex bolt). 4 Using the 5/16 Hex key, turn the sample block locking bolt clockwise until it is flush with the locking bar. 5 Again, press on the right and left sides of the front surface of the sample block to ensure that it is seated securely. 6 Replace the thermal cycler access cover as follows: GR2025b 2 a. Fit the lip at the bottom of the access cover over the lower edge of the bay. GR2023b b. Push the cover towards the instrument until it snaps into place. System Maintenance 7-7 To replace the sample block: Step Action If using a plate handler, replace the covers for the fixed-position bar code reader and the underlying platform (removed in step 6 on page 7-5). GR2009 7 (continued) Fixed-position bar code reader and underlying platform covers 8 Plug in and turn on the 7900HT instrument. 9 Confirm the function of the installed sample block module as follows: a. Launch the Automation Controller Software. b. Click the Thermal Status tab. Does the software display temperatures? Then… Yes the installation is successful. The presence of temperature readings confirm that the 7900HT instrument successfully established the connection to the new sample block. No the 7900HT instrument is unable to establish communication with the new sample block. To troubleshoot the problem: a. Turn off and unplug the 7900HT instrument. b. Remove the thermal cycler access cover. c. Press on the right and left sides of the front plate of the sample block to ensure that it is seated securely. d. Reinstall the thermal cycler access cover. e. Repeat step 8 until you hear a high-pitched tone confirming communication between the instrument and sample block. 10 Once the sample block is loaded into the instrument do the following: a. Perform a background run (see page 7-13) to verify that the sample block: – Is connected and working properly – Contains no contaminants that will interfere with fluorescent detection b. If changing sample block formats, perform any remaining tasks outlined in the “Sample Block Installation Checklist” on page 7-4. 7-8 System Maintenance Changing the 7900HT Plate Adapter When to Perform Remove and replace the 7900HT instrument plate adapter after changing the sample block module format (for example, replacing a 384-well sample block module with a 96-well version). Note The sample block must be used with the corresponding plate adapter of the same plate format. Materials Required The procedure below requires the use of the following materials: Material Part Number One of the following: See page D-3 ♦ 384-Well Plate Adapter ♦ 96-Well Plate Adapter 3/32 inch Hex key — Changing the Plate To replace the 7900HT instrument plate adapter: Adapter Step 1 Action If the instrument tray is inside the 7900HT instrument, move the instrument tray to the OUT position as follows: a. Launch the SDS software. b. From the File menu, select New. The New Document dialog box appears. c. Click OK. The software generates a plate document. d. Click the Instrument tab. e. From the Real-Time tab of the Instrument tabbed page, click Open/Close. The instrument tray rotates to the OUT position. f. From the File menu, select Exit. The SDS software exits. 2 Remove the four screws attaching the plate holder to the plate arm. Unscrew 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A B C D E F G H I J K L M N O P Unscrew 3 Remove the plate adapter from the instrument tray. Note If changing sample block formats (for example, replacing a 384-well sample block with a 96-well version), store the plate adapter with the sample block module of the same format. System Maintenance 7-9 To replace the 7900HT instrument plate adapter: Step 4 (continued) Action Place the new plate adapter into the instrument tray with the A1 label in the rear-left corner (see below). IMPORTANT Make sure to install the correct version of the plate adapter (384- or 96-well) for the plate format you intend to use. The plate adapters are labeled for the consumable format they support. Well A1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A B C D E F G H I J K L M N O P 5 Label (384- or 96-well) Replace and tighten the four screws in the order shown below: 3 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A B C D E F G H I J K L M N O P 2 4 IMPORTANT The order in which the screws are tightened is important to ensure proper alignment of the plate to the sample block within the 7900HT instrument. 7-10 System Maintenance Decontaminating the Sample Block When to Perform The following procedure describes how to decontaminate the wells of a sample block module. The procedure will eliminate residual PCR related products, including fluorescent labeled TaqMan® probes. Clean the sample block as often as needed. IMPORTANT If preforming a cleaning or decontamination method other than the one in this manual, check with Applied Biosystems first to ensure that the method will not damage the sample block module or the 7900HT instrument. Materials Required The cleaning procedure requires the following materials: Material Part Number Cotton swabs — 10% Sodium hypochlorite (bleach) solution — Isopropanol, 100 percent pure — 5/32 in Hex key — ! WARNING CHEMICAL HAZARD. Sodium hypochlorite (bleach) is a liquid disinfectant that can be corrosive to the skin and can cause skin depigmentation. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. ! WARNING CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. System Maintenance 7-11 Cleaning the Sample To clean the sample block wells: Block Wells Step 1 Action Identify the contaminated wells as follows: a. If not already run, perform a background run as explained on page 7-13. b. Identify the contaminated wells on the sample block by following the procedure, “Isolating Sample Block Contamination” on page 8-8. 2 Remove the sample block from the 7900HT instrument as explained in “Removing the Sample Block” on page 7-5. 3 Using the following figure as a guide, locate the suspected contaminated wells on the sample block. Circuitry and connections to the instrument (Do Not Touch) Warning GR2028 Well A-1 4 Pipet the appropriate volume of 10% bleach solution into each suspected contaminated well of the sample block module. ♦ For a 96-well sample block, pipet 150 µL bleach solution to each well. ♦ For a 384-well sample block, pipet 40 µL bleach solution to each well. 5 Allow the sample block to sit for 3-5 min. 6 Using a pipet, remove the bleach solution from the wells of the sample block. 7 Rinse (pipet and remove) each contaminated well with 3 treatments of deionized water at the appropriate volume for the sample block. ♦ For a 96-well sample block, rinse affected wells with 150 µL deionized water. ♦ For a 384-well sample block, rinse affected wells with 40 µL deionized water. Note 7-12 System Maintenance Absolute isopropanol can be substituted for water in the third treatment. 8 Remove any remaining isopropanol or water from the wells of the sample block module. 9 Replace the sample block as explained in “Replacing the Sample Block” on page 7-7. 10 Run a background plate to confirm that the contamination has been removed. Performing a Background Run When to Perform Applied Biosystems recommends running a background plate weekly or as often as necessary depending on instrument use. Purpose of A background run measures the ambient fluorescence in a background plate Background Runs containing deionized water. During the run, the 7900HT instrument conducts a continuous scan of the plate for 2 minutes at 60 °C. Afterwards, the SDS software averages the spectrum recorded during the run and is used to extract the resulting spectral component to a calibration file. The software uses the calibration file during subsequent runs to remove the background signal from the run data. Because the background signal can change with instrument age, Applied Biosystems recommends regenerating the Background component calibration every month. Note Background runs can also be used to detect and troubleshoot sample block contamination. About the Fluorescence collected by the ABI PRISM 7900HT Sequence Detection System Background includes a “background” component, a fluorescent signal that is inherent to the Component system. The background component is a composite signal found in all spectral data that consists of fluorescence from several sources including: the background electronic signal, the sample block, the water within the consumable, and the plastic consumable itself. Because the background signal can interfere with the precision of SDS data, the 7900HT instrument has been engineered to minimize the background signal. Additionally, the SDS software also algorithmically eliminates the background signal from each fluorescent sample to maximize the instrument’s sensitivity (see page A-5). Materials Required The following materials are required to perform a background run: Material Part Number One of the following: ♦ Background Plate included in a ABI PRISM Sequence Detection Systems Spectral Calibration Kit, or ♦ Make a background plate (requires the following) – ABI PRISM® See page D-4 Optical 394- or 96-Well Reaction Plate – ABI PRISM® Optical Adhesive Cover or ABI PRISM Optical Flat Caps – Pipettor, 100-µL (with pipet tips) Centrifuge, with plate adapter — System Maintenance 7-13 Prepare a To prepare a plate document for the background run: Background Plate Step Action Document 1 Launch the SDS software. 2 From the File menu, select New. The New Document dialog appears. 3 Configure the New Document dialog box as follows: Drop-Down List Select Assay Background Container <the appropriate plate format> Template Blank Template 4 If the background plate is labelled with a bar code, click the Barcode text field and scan the bar code number using the hand-held bar code reader. 5 Click OK. The software creates a plate document with the attributes for a background run. Note Do not modify the background plate document. The method for a Background run is hard-coded into the SDS software and consists of a single hold at 60 °C for 2 min. Because the plate contains only deionized water, the plate document does not require sample or detector labels. 6 Save the plate document as follows: a. From the File menu, select Save. The Save dialog appears. b. Click the File name text field, and type Background_<date in MMDDYY format>. For example, the file name for a background plate run on May 31, 2001 would be: Background_053101 c. Click Save. The software saves the plate document. The software is now configured for the Background run. 7 7-14 System Maintenance Prepare and run the plate as explained on page 7-15. Preparing and To conduct the background run: Running a Step Action Background Plate 1 Choose from the following: If using… Then… a background plate from a Spectral Calibration Kit Remove the plate from the freezer and allow it to thaw to room temperature. an ABI PRISM Optical Reaction Plate a. Pipet deionized water to each well of the plate. – If using a 384-well plate, add 20 µL per well. – If using a 96-well plate, add 50 µL per well. b. Seal the plate using an optical adhesive cover or optical flat caps. 2 Briefly centrifuge the background plate. 3 Load the background plate into the 7900HT instrument as follows: a. From the plate document in the SDS software, click the Instrument tab. b. From the lower portion tab, click the Real-Time tab. c. From the Real-Time tab of the Instrument tabbed page, click Open/Close. d. Place the background plate into the instrument tray as shown below. Well A1 PECY001DL3 Note 4 Position the plate so that the bar code faces towards the front of the instrument The A1 position is located in the top-left side of the instrument tray. Click Start. The 7900HT instrument begins the background run. Note Before starting the run, the instrument may pause (up to 15 min) to heat the heated cover to the appropriate temperature. 5 When the background run is complete and the Run Complete dialog box appears: a. Click OK to close the dialog box. b. Click Open/Close, and remove the background plate from the instrument tray. c. Extract the background component as explained on page 7-16. System Maintenance 7-15 Extracting the In this procedure you will extract the calibration values from the background plate Background document. Once extracted, the SDS software stores the data as part of the calibration file located in the Calibration subdirectory of the SDS 2.0 directory. To extract the background component from the run data: Step 1 Action From the Analysis menu, select Extract Background. The software attempts to extract the background signal and displays the success of the extraction in a dialog box. Dialog Box Then... the run is successful The raw spectra read from the Background plate conform to acceptable limits. Proceed to step 3. the run is unsuccessful. The software has stopped the extraction because one or more raw spectra exceed 2500 FSU. Troubleshoot and decontaminate the sample block as explained in “Background Runs” on page 8-8. 2 From the File menu, select Save. The software saves the plate document. 3 From the File menu, select Close. The software closes the plate document. 7-16 System Maintenance Performing a Pure Dye Run When to Perform Applied Biosystems recommends performing spectral calibration: ♦ Every 6 months depending on instrument use ♦ After changing sample block formats (see page 7-4) IMPORTANT Always run a background plate before performing a Pure Dye calibration. Purpose of Pure Dye Pure dye data is generated from the results of a pure dye run in which the SDS Runs software collects spectral data from a set of dye standards during a 2-min hold at 60 °C. The software stores the spectral information for the pure dye standards within a calibration file located in the SDS directory. After the run, the software extracts each component dye spectrum from the collected data in the pure spectra run file. IMPORTANT Because the age and use of instrument components can affect pure spectra readings, Applied Biosystems recommends updating the pure spectra data files once or twice annually depending on instrument use. Components of the The ABI PRISM 7900HT Sequence Detection System monitors fluorescent signals Pure Dye Spectra generated by several dyes FAM™, NED™, ROX™, SYBR®, TAMRA™, TET™, and VIC™. The figure below compares the pure spectra for each dye. 1 2 3 4 5 6 7 Dye Peak (nm) 1 FAM 2 SYBR ~520 3 TET ~540 VIC ~550 JOE ~550 5 NED ~570 6 TAMRA ~580 7 ROX ~610 4 ~520 Note The 7900HT instrument supports the detection of custom pure dyes (dyes other than those provided by Applied Biosystems). To add custom pure dyes to the Pure Dye set for your instrument, see “Adding Custom Dyes to the Pure Dye Set” on page 7-21. After a run, the SDS software receives run data in the form of a raw spectra signal for each reading. To make sense of the raw data, the software must determine the contribution of each fluorescent dye used in the sample through a process called multicomponenting (see page A-5). The software accomplishes the separation by comparing the raw spectra with a set of pure dye standards contained within a calibration file. When a plate document is saved after analysis, the software stores the pure spectra information with the collected fluorescent data for that experiment. System Maintenance 7-17 Materials Required The following materials are required to perform a pure dye run: Material Part Number Sequence Detection Systems Spectral Calibration Kit 384-Well Version See page D-3 96-Well Version Pure Dye Platea,b Product — Insertb — Centrifuge, with plate adapter — a. The 96-Well version of the Spectral Calibration Kit contains 2 Pure Dye plates. b. Included with the Sequence Detection Systems Spectral Calibration Kit. Preparing a IMPORTANT A background run must be performed prior to running a pure dye plate. See Pure Dye Plate “Performing a Background Run” on page 7-13 for more information. Document To prepare a plate document for the pure dye run: Step Action 1 Remove the pure dye plate from the freezer, place it aside to thaw to room temperature, and return to the computer. 2 Launch the SDS software. 3 From the File menu, select New. The New Document dialog appears. 4 Select the following options from the menus within the New Document dialog box. From… Select… Assay Pure Dyes Container <Select the appropriate plate format> Template ♦ For a 384-Well Pure Dye Run, select 384 Well Pure Dyes Plate.sdt. ♦ For 96-Well Pure Dye Runs, select the template matching the Pure Dye plate you intend to run. – To run Plate 1 (containing FAM, JOE, NED, and ROX), select 96 Well Pure Dyes Plate 1.sdt. – To run Plate 2 (containing SYBR, TAMRA, TET, and VIC), select 96 Well Pure Dyes Plate 2.sdt. Note If no templates are available, construct a Pure Dye plate document using the product insert from the Sequence Detection Systems Spectral Calibration Kit and the procedure on page 7-22. 5 Remove the pure dye plate from its packaging. 6 Click the Barcode text field, and scan the bar code number using the hand-held bar code reader. 7 Click OK. The software displays a plate document with the attributes for a pure dye run. 7-18 System Maintenance To prepare a plate document for the pure dye run: Step 8 (continued) Action Save the Pure Dye plate document as follows: a. From the File menu, select Save. b. From the Files of type drop-down list, select ABI PRISM SDS Single Plate (*.sds). c. From the Save dialog, click the File name text field, and choose from the following: Plate Format Type… 384-Well PureDye_<date in MMDDYY format> For example, the file name for a plate run on May 31, 2001 would be: PureDye_053101. 96-Well PureDye_Plate<plate number>_<date in MMDDYY format> For example, the file name for a Pure Dye Plate 1 run on May 31, 2001 would be: PureDye_Plate1_053101. d. Click Save. The software saves the plate document. 9 Prepare and run the pure dye plate as explained below. Preparing and IMPORTANT A background run must be performed prior to running a pure dye plate. See Running the “Performing a Background Run” on page 7-13 for more information. Pure Dye Plate To prepare the pure dye run: Step Action 1 Briefly centrifuge the pure dye plate. 2 Load the pure dye plate into the 7900HT instrument as follows: a. From the plate document in the SDS software, click the Instrument tab. b. From the Real-Time tab in the Instrument tabbed page, click Open/Close. The instrument tray rotates to the OUT position. c. Place the pure dye plate into the instrument tray. Note 3 The A1 position is located in the top-left side of the instrument tray. Click Start. The 7900HT instrument begins the pure dye run. The method for a pure dye run is hard-coded into the software and consists of a single 2-min hold at 60 °C. Note Before starting the run, the instrument may pause (up to 15 min) to heat the heated cover to the appropriate temperature. 4 When the pure dye run is complete and the Run Complete dialog box appears: a. Click OK to close the dialog box. b. Click Open/Close, and remove the pure dye plate from the instrument tray. c. Extract the pure dye calibration information as explained on page 7-20. System Maintenance 7-19 Extracting Pure Dye The purpose of viewing the data in the Pure Dye Wizard is to eliminate irregular pure Information from dye peaks from the data set. The wizard presents the spectral data from the pure dye the Analyzed Run plate in sets of three wells, each containing the same pure dye. Because the wells displayed by the wizard contain the pure dye at an identical concentration, the signal peaks for the set should be identical. Occasionally, pipetting inaccuracies or contamination can cause a well signal to shift slightly. While viewing the data, the outlying peaks must be eliminated. To extract the pure dye information from the run data: Step 1 Action From the Analysis menu, select Extract Pure Dye Wizard. The Extract Pure Dye Wizard dialog appears. 2 Follow the instructions as explained by the Extract Pure Dye Wizard to extract the pure dye spectra. When presented with each screen, do the following: a. Inspect the spectra for shifts in peak location. Wavelength shift Click here to remove it b. If the data set contains a outlying peak, eliminate it by clicking check box of the associated well. Note Dye spectra are generally acceptable if they peak at the same location as their group but diverge slightly at other wavelengths. c. Click Next to view the next three wells. d. Repeat steps a to c for all remaining wells until prompted with a message reporting the extraction of the pure dyes. The software extracts the pure spectra and stores the data as a component of the calibration file. 3 From the File menu, select Save. The software saves the plate document. 4 From the File menu, select Close. The software closes the plate document. 5 7-20 System Maintenance If performing spectral calibration of a 96-well block module, repeat the previous procedures on pages 7-18, 7-19, and 7-20 to run the second Pure Dye plate. Adding Custom Dyes to the Pure Dye Set When to Perform The ABI PRISM 7900HT Sequence Detection System can be used to run assays designed with custom dyes (dyes not manufactured by Applied Biosystems). However, before using custom dyes with the 7900HT instrument, you must create and run a custom pure dye plate. Materials Required The following materials are required to perform a pure dye run: Material Part Number ABI PRISM Optical 394- or 96-Well Reaction Platea See page D-3 ABI PRISM Optical Adhesive Cover or ABI PRISM Optical Flat Caps Custom Dye(s) — Pipettor, 100-µL (with pipet tips)a — Centrifuge, with plate adaptor — a. Used to add custom dyes to the Dye Set. Creating a Custom Note The purpose of the custom pure dye plate is identical to that of an ABI PRISM Pure Dye Pure Dye Plate Plate. The SDS software uses the custom plate to create a spectral standard for multicomponenting the custom dye. To create a pure dye plate for custom dyes: Step Action 1 Prepare a microplate with a dilution series of the custom dye. 2 Launch the SDS software. 3 Create an allelic discrimination plate document and run the dilution series plate. Note It is not necessary to configure detector, sample, and method information for the dilution series plate document. The purpose of the run is to establish the correct working concentration for the dye by viewing the intensity of the raw spectra produced by the wells in the dilution series. 4 From the Analysis menu, select Analyze. The software analyzes the raw run data, 5 Click the Show Raw Data Plot button ( ) from the Display toolbar. The software displays the Raw Data Plot. 6 From the raw spectra, determine the highest concentration of dye that does not produce a saturated signal, and record it for future use. Note Saturated signals are characterized by their high peaks that rise beyond detectable levels (> 65,000 fluorescent units) and appear as plateaus on the Raw Data plot. The concentration of the custom dye that yields the highest possible signal but does not saturate is the maximum concentration for use with the 7900HT instrument. 7 Repeat steps 1 to 6 for any additional custom dyes. System Maintenance 7-21 To create a pure dye plate for custom dyes: GR2107 Custom Dye 8 Custom Dye 7 Custom Dye 6 Custom Dye 5 Custom Dye 4 Custom Dye 3 Create a pure dye plate for the custom dye(s) by pipetting each custom dye to at least three columns of an ABI PRISM® Optical Reaction Plate at the concentrations determined in step 7. Custom Dye 1 8 Action Custom Dye 2 Step (continued) IMPORTANT The optical configuration of the 7900HT instrument requires that each pure dye occupy at least 3 columns of the Pure Dye plate to permit adequate data collection. 9 Seal the plate using an optical adhesive cover or optical flat caps. 10 Create a template document for the custom pure dye plate as explained below. Constructing a To create a template for running the custom pure dye plate: Custom Pure Dye Step Action Plate Document 1 Launch the SDS software. Template 2 Add the new dye to the software using the Dye Manager as follows: a. From the Tools menu, select Dye Manager. The Dye Manager dialog box appears. b. Click Add. The Add Dye dialog box appears. c. Type a name for the custom dye, and click OK. The software adds the new dye to the Custom dye list. d. Repeat steps 2 and 3 to add any additional custom dyes to the Dye Manager. e. Click Done. The SDS software makes the new dyes available to pure dye plate documents. 3 Create a custom pure dye plate document for the run as follows: a. From the File menu, select New. The New Document dialog box appears. b. Configure the drop-down lists with the following options: Drop-Down List Select Assay Pure Dyes Container <the appropriate plate format> Template Blank Template c. Click OK. The software creates a new plate document. 7-22 System Maintenance To create a template for running the custom pure dye plate: Step 4 (continued) Action Apply pure dyes to the custom plate document as follows: a. Select the wells containing the custom dye. b. From the Setup tabbed page, click the Dyes drop-down list, and select the appropriate dye. The software applies the dye to the selected wells. c. Repeat steps 6 and 7 to configure the plate document with any additional custom dyes. Dyes drop-down list Custom dye added to selected wells of the plate document 5 Save the custom pure dye plate document as a template file as follows: a. From the File menu, select Save. The Save dialog appears. b. Navigate to Program Files > Applied Biosystems > SDS 2.0 > Templates. The Templates directory appears within the Look in text field. By saving the template to the Templates folder, it becomes available from the Template drop-down list in the New Document dialog box. c. From the Files of type drop-down list, select ABI PRISM SDS Template Document. d. Click the File name text field, and type a name for the template document. e. Click Save. The software saves the plate document as a template file (*.sdt). 6 Run the custom pure dye plate as explained in “Preparing and Running the Pure Dye Plate” on page 7-19. System Maintenance 7-23 Verifying Instrument Performance Using a TaqMan RNase P Plate When to Perform Applied Biosystems recommends running a TaqMan® RNase P Instrument Verification Plate: ♦ When changing sample block formats for the first time ♦ As needed to verify the function of the 7900HT instrument Purpose of TaqMan RNase P Instrument Verification Plate is an experiment run to verify the RNase P Runs performance of the 7900HT instrument. The sealed plate is pre-loaded with the reagents necessary for the detection and quantification of genomic copies of the human RNase P gene (a single-copy gene encoding the moiety of the RNase P enzyme). Each well contains pre-loaded reaction mix (1X TaqMan® Universal PCR Master Mix, RNase P primers, and FAM-labeled probe) and template. The following figures illustrate the arrangement of standards and samples on the RNase P plate. As shown below, the RNase P plate consists of 5 columns of template standards (1250, 2500, 5000, 10,000, and 20,000 copies) and two unknown populations (5000 and 10,000 copies). RNase P Plate Sample Configuration Unknown 2 10000 GR2107 Unknown 1 5000 NTC STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 384-Well 96-Well Unknown 1 5000 NTC STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 Unknown 1 10000 7-24 System Maintenance Materials Required The following materials are required to perform the RNase P run: Material Part Number TaqMan RNase P Instrument Verification Plate See page D-4 Centrifuge, with plate adaptor Preparing a RNase P To prepare a plate document for the RNase P plate: Plate Document Step Action 1 Remove the TaqMan RNase P Instrument Verification Plate from the freezer and allow it to thaw to room temperature. 2 Launch the SDS software. 3 From the File menu, select New. The New Document dialog appears. 4 5 Configure the New Document dialog box as follows. Drop-Down List Select Assay Absolute Quantification Container <the appropriate plate format> Template <the template file of the appropriate plate format> If desired, enter the bar code information into the plate document as follows: a. Click the Barcode text field. b. Remove the RNase P plate from the packaging and scan its bar code using the hand-held bar code reader. 6 Click OK. The software creates a plate document. Note Do not modify the RNase P plate document. The template is pre-programmed with detector and method information for the run. 7 Save the plate document as follows: a. From the File menu, select Save. The Save dialog appears. b. Click the Barcode text field and either: – Type a name or bar code number for the plate, and click Save. – Using the hand-held bar code reader, scan the bar code number. c. From the Files of type drop-down list, select ABI PRISM SDS Single Plate (*.sds). d. Click Save. The software saves the plate document. The software is now configured for the RNase P run. 8 Prepare and run the RNase P plate as explained on page 7-26. System Maintenance 7-25 Preparing and To run the RNase P plate: Running an Step Action RNase P Plate 1 Briefly centrifuge the TaqMan RNase P Instrument Verification Plate. 2 From the plate document in the SDS software, click the Instrument tab. The software displays the Instrument tabbed page. 3 From the lower portion of the Instrument tab, click the Real-Time tab. The software displays the Real-Time tabbed page. 4 If the instrument tray is within the 7900HT instrument, click Open/Close. The instrument tray rotates to the OUT position. 5 Place the RNase P plate into the instrument tray. Note 6 The A1 position is located in the top-left corner of the instrument tray. Click Start. The 7900HT instrument begins the run. Note Before starting the PCR run, the instrument may pause (up to 15 min) to heat the heated cover to the appropriate temperature. 7 When the run is complete: a. Analyze the run data as explained on page 6-9. a. Set the baseline and threshold values for the analyzed data as explained on page 6-10. b. Verify the performance of the 7900HT instrument as explained below. Verifying The install specification of the ABI PRISM 7900HT Sequence Detection System Instrument demonstrates the ability to distinguish between 5,000 and 10,000 genome equivalents Performance with a 99.7% confidence level for a subsequent sample run in a single well. The following equation verifies the 7900HT install specifications: [ ( CopyUnk 1 ) – 3 ( σCopyUnk1 ) ] > [ ( CopyUnk 2 ) – 3 ( σCopyUnk2 ) ] where: CopyUnk1a σCopyUnk1a = Average Copy Number of Unknown #1 (10,000 replicate population) = Standard Deviation of Unknown #1 (10,000 replicate population) CopyUnk1 a = Average Copy Number of Unknown #2 (5000 replicate population) σCopyUnk2 a = Standard Deviation of Unknown #2 (5000 replicate population) a. These values can easily be obtained from the experimental report window. Note Up to 6 wells from each replicate group in a 96-well TaqMan RNase P Instrument Verification Plate can be ignored to meet specification. Note Up to 10 wells from each replicate group in a 384-well TaqMan RNase P Instrument Verification Plate can be ignored to meet specification. 7-26 System Maintenance Section: Maintaining the Plate Handler In This Section This section contains the following information: Topic See Page Adjusting the Sensitivity of the Plate Sensor Switch 7-28 Aligning the Plate Handler 7-32 Aligning the Fixed-Position Bar Code Reader 7-40 Cleaning and Replacing Gripper Finger Pads 7-46 Automation Module Refer to the figure below for the components discussed in this section. Components GR2014a Adjustment knob Gripper Plate-sensor switch (cross-sectional view of the gripper) Plate stack Expansion stacks Zymark Twister Microplate Handler Fixed-position bar code reader Plate Stack Positions The Zymark Twister Microplate Handler alignment is performed using the Zymark® Twister Software. The software refers to the positions of the plate stacks differently than the Automation Controller Software. The following diagram lists the positions defined by the Zymark Twister Software and the Automation Controller equivalents. Zymark Twister Software Automation Controller Position 0 Output Position 1 (unused) Position 2 Instrument Position 3 (unused) Position 4 Stack 1 Position 5 Stack 2 Bar code Position 6 Stack 3 Well A1 Position 7 Stack 4 0 7 1 2 6 3 5 4 (front of instrument) System Maintenance 7-27 Adjusting the Sensitivity of the Plate Sensor Switch When to Perform The plate sensor switch located underneath the arm of the Zymark Twister Microplate Handler requires adjustment under the following circumstances: ♦ When changing sample block module formats ♦ If the plate handler is having difficulty sensing plates Materials Required The following materials are required to adjust the plate sensor switch: Material Part Number ABI PRISM Optical Reaction Plate (of the current sample block format) See page D-1 Adjusting the Switch The dimensions of different plate formats can place different requirements on how the plate handler grips plates. To ensure smooth operation of the automation accessory, adjust the plate sensor switch when changing consumable formats. To adjust the plate sensor switch: Step 1 Action Turn off the Zymark Twister Microplate Handler. ! WARNING PHYSICAL HAZARD. The Zymark Twister Microplate Handler must be turned off at all times during the following procedure. Failure to comply can result in physical injury to the user or damage to the plate handler. 2 Clear the switch position by turning the thumb wheel all the way to the Up extreme (as indicated on the side panel). GR2014a Thumb wheel Plate-sensor switch 3 Begin the adjustment of the sensor switch as follows: a. Grasp a 96- or 384-well ABI PRISM Optical Reaction Plate by the sides making sure not to place pressure in the center of the plate to deform it. b. Place the plate between the fingers of the gripper assembly and align it to the middle of the centering device. c. While holding the plate in position, slowly turn the thumb wheel to lower the switch onto the reaction plate until the switch: – Contacts the top of the plate, and – Emits a soft, audible “clicking” noise IMPORTANT The sound emitted by the sensor switch is very faint and may be difficult to hear. To make the adjustment easier, place your ear close to the sensor switch while making the adjustment and listen for the switch to engage. 7-28 System Maintenance To adjust the plate sensor switch: Step 4 (continued) Action Remove the plate and listen for the plate-sensor switch to disengage. Did you hear the switch disengage? No Then… a. Move the switch Down a few steps by turning the thumb wheel in the direction indicated on the arm. b. Replace the plate within the gripper and listen for the switch to engage: – If you do not hear the switch engage, then remove the plate and repeat steps a and b above. – If you hear the switch engage, remove the plate and continue to step a below. Yes a. Move the switch Up by turning the thumb wheel one step in the direction indicated on the arm. b. Replace the plate and listen for the switch to engage: – If you hear the switch engage, remove the plate and repeat steps a and b. – If you do not hear the switch engage, then you have successfully identified the zero point of the plate-sensor switch. Note At the zero point, one step of the thumb wheel in the Down direction causes the switch to engage. 5 Once the zero point is established, carefully turn the thumb wheel in the Down direction the number of steps appropriate for your plate format as indicated below: Plate Format Turn the thumb wheel in the Down direction… 96-Well 20 steps 384-Well 15 steps Note If you lose count, begin again from step 4 and identify the zero point for the switch. 6 Test the adjustment as explained on page 7-30. System Maintenance 7-29 Testing the To test the sensitivity of the plate sensor switch: Adjustment Step Action 1 Place the reaction plate in the input stack 1 of the plate handler. 2 Turn on the 7900HT instrument, the plate handler, and the computer. 3 From the Start menu, select Programs > Zymark Twister Plate Handler > Twister. The Zymark Twister Software launches. 4 Click Manual Control. The software displays the Manual Control dialog box. 5 Click stack 4. Click The plate handler arm moves over the input stack. 6 Click Find Plate. If the adjustment was successful, the plate handler arm will lower upon the plate until the plate detector switch engages confirming the presence of the plate. If plate handler arm emits a grinding sound, adjust the plate sensor switch as follows: a. From the Zymark Twister Software, click Vertical Home to raise the plate handler arm. b. Turn the thumb screw in the Down direction 10 steps. c. Repeat step 6 until the plate handler arm successfully detects the plate. 7 Click Close Gripper, then click Vertical Home. If the adjustment was successful, the plate handler arm will grasp the plate and remove it from the plate stack. If plate handler arm stops before the gripper fingers are able to contact the plate and fails to grasp or pick up the plate, adjust the plate sensor switch as follows: a. Turn the thumb screw in the Up direction 10 steps. b. Grasp the plate with one hand and, from the Zymark Twister Software, click Open Gripper to release the plate. c. Replace the reaction plate into input stack 1 of the plate handler. d. Repeat steps 6 and 7 until the plate handler arm successfully retrieves the plate. 8 Grasp the plate with one hand and, from the Zymark Twister Software, click Open Gripper to release the plate. 7-30 System Maintenance To test the sensitivity of the plate sensor switch: Step 9 (continued) Action Exit the Zymark Twister Software. a. Click Main Menu. The software displays the main menu. b. Click Quit Application. The software closes. 10 Note A bug in the Zymark Twister Software can cause portions of the program to persist in memory even after the software has been closed. Because the Zymark Twister Software conflicts with the SDS software, the residual elements of the software must be closed within the Windows Task Manager before continuing. Confirm that the stack has closed by viewing the Task Manager. a. Press the Crtl + Alt + Del keys in unison. The Windows Security dialog box appears. b. Click Task Manager. The Task Manager dialog box appears. c. Confirm that the software has closed by looking for the Zymark Twister Software entry in the Task list. If the software is still running, click the software entry and click Close to exit the remaining software. d. From the File menu, select Exit. System Maintenance 7-31 Aligning the Plate Handler When to Perform Perform the following procedure if the ABI PRISM 7900HT Sequence Detection System is moved or the Zymark Twister Microplate Handler becomes mis-aligned. Symptoms that the plate handler is out of alignment include: ♦ Excessive downward movement of the plate handler arm (the arm grinds when grasping or releasing plates) ♦ The plate handler arm collides with the plate stacks ♦ The plate handler arm releases plates above the bottom of the plate stacks ♦ Reaction plates tip or tilt when placed into the instrument tray by the arm Preparing the To move the instrument tray to the OUT position: Instrument for the Step Action Alignment Remove the covers for the fixed-position bar code reader and the underlying platform. GR2009 1 2 Fixed-position bar code reader and underlying platform covers Loosen the three black thumb screws on the platform connecting the 7900HT instrument and the plate handler base. Black thumb screws 3 Move the instrument tray to the out position. a. Launch the Automation Controller Software. If an error dialog appears reading, ‘Machine calibration values are not valid. Please refer to documentation for calibration process,’ click OK. b. Click Open/Close ( ). The 7900HT instrument moves the instrument tray to the out position, perpendicular to the instrument. c. From the File menu, select Exit. The software quits the Automation Controller Software. 4 From the Start menu, select Programs > Zymark Twister Plate Handler > Twister. The Zymark Twister Software launches. 5 Click Manual Control. The software displays the Manual Control dialog box. 7-32 System Maintenance Aligning The alignment of input stack 1 (position 4 in the Zymark Twister Software) is the first Input Stack 1 step in the alignment procedure. This alignment provides the basis for aligning all (Zymark Position 4) subsequent stacks on the plate handler. To align the input stack 1 (Zymark position 4): Step Action 1 Place an empty plate into input stack 1(Zymark position 4). 2 From the Zymark Twister Software, click position 4. Click The plate handler arm moves over the input stack. 3 Using the Vertical Positioning commands, lower the plate handler arm until it is just above the stack. The Vertical Positioning box offers four ways to move the plate handler arm: ♦ Move the slider for large increments. ♦ Click inside the slider bar to move the arm in 250 step increments. ♦ Click the lower arrow on the bar to move the arm in 50 step increments. ♦ Click the up or down arrows in the Vertical Adjustment text box to move the arm in 1 step increments. ♦ Click the Vertical Adjustment text box, type a value, and press Enter to move the arm into a specific location. Slider Slider bar (250 steps per click) Down arrow (50 steps per click) Text box arrows (1 step per click) 4 Check the rotary position of the plate handler arm to confirm that the gripper: ♦ is centered over the stack ♦ will not contact the sides of the stack when lowered System Maintenance 7-33 To align the input stack 1 (Zymark position 4): Step 5 (continued) Action Using the Rotary Adjustment arrows, adjust the rotational position of the gripper so that it is centered over the input stack and will not contact the sides when lowered. ♦ To move the plate handler arm clockwise, click the up arrow. ♦ To move the plate handler arm counter-clockwise, click the down arrow. Up arrow (moves the arm clockwise) Down arrow (moves the arm counter-clockwise) 6 Using the Vertical Positioning commands, carefully lower the plate handler arm into the stack. Adjust the Rotary Adjustment value as needed to center the gripper within the stack. 7 Once the gripper is centered within the stack, click Find Plate. The plate handler arm lowers upon the plate. Confirm the following: ♦ the plate is in the middle of the gripper span ♦ the plate sensor switch is contacting the plate ♦ the gripper or plate do not contact the side of the stack 8 Click Close Gripper. The gripper grips the plate between its fingers. 9 Select Vertical Home. The plate handler raises the arm to its highest position. If the plate contacts the sides of the stack, re-adjust the rotary position of the plate handler arm until the plate moves freely in the stack. Note Contact between the plate and the stack or all stacks may be unavoidable. However, try to minimize the contact as much as possible. 10 Using the Vertical Positioning commands, raise and lower the plate handler arm several times to check the alignment. 11 Lower the plate handler arm to the bottom of the plate stack, click Rotary Offset, and click Yes. The software records the rotary position for the Zymark position 4 (input stack 1). 12 Click Open Gripper. The gripper releases the plate. 7-34 System Maintenance Aligning the Plate The next step is to align the plate handler arm to the instrument tray (Zymark Handler to the position 2). This alignment will ensure a smooth exchange between the plate handler Instrument arm and the instrument tray during operation of the instrument. Note The following procedure requires you to position the plate handler relative to the 7900HT instrument, Before moving the plate handler, loosen the three black thumb screws on the platform connecting the 7900HT instrument and the plate handler. To adjust the plate handler relative to the 7900HT instrument: Step 1 Action If not already present, place an empty plate into input stack 1(Zymark position 4) and pick it up with the plate handler arm as follows: a. From the Zymark Twister Software, click position 4. b. Click Find Plate. c. Click Close Gripper. 2 Click position 2. Click here The plate handler arm moves over the instrument tray. 3 Use Vertical Positioning to lower the plate handler arm until it is approximately 1 cm above the instrument tray. 4 Using the Rotary Adjustment arrows, center the gripper and plate along the Y-axis of the instrument tray. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A B C D E F Center the plate G H I J K L M N O P 5 Center the gripper and plate along the X-axis of the instrument tray by sliding the plate handler and base towards or away from the 7900HT instrument. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A B C D E F G H I J K L M N O Center the plate 6 GR2012 P Again, using the software to move the plate handler arm, center the gripper and plate along the Y-axis of the instrument tray as explained in step 4. System Maintenance 7-35 To adjust the plate handler relative to the 7900HT instrument: Step (continued) Action 7 Using the Vertical Positioning commands, carefully lower the plate handler arm onto the instrument tray and confirm that the plate rests squarely within it. 8 Tighten the three black thumb screws on the platform connecting the 7900HT instrument and the plate handler. 9 Release the plate from the plate handler arm. a. Click Open Gripper. b. Click Vertical Home. 10 Click Find Plate. The plate handler arm lowers onto the plate. 11 Save the rotary and vertical offset information: a. Click Rotary Offset, and click Yes. The software records the rotary position for the plate drawer (Zymark position 2). b. Click Vertical Offset, and click Yes. The software records the vertical position for the plate drawer. Re-checking the Now that the positions of the plate handler and instrument are fixed, the plate handler Input Stack 1 stacks can be aligned and the positional values recorded. To re-check the position of input stack 1 (Zymark position 4): Step Action 1 Place an empty plate into input stack 1. 2 From the Zymark Twister Software, click the icon for stack 4. The plate handler arm moves over the input stack. 3 Using the Vertical Positioning commands, lower the plate handler arm until it is 1 cm above the stack and verify that it is centered on the stack. If necessary, center the stack using the Rotary Adjustment arrows. 4 Carefully lower the plate handler arm into the stack. Center the gripper as it moves down the stack by adjusting the Rotary Adjustment arrows if needed. 5 Once the plate handler arm is centered within the stack, click Find Plate. The plate handler arm lowers upon the plate. Confirm the following: ♦ The plate is in the middle of the gripper span. ♦ The plate sensor switch is contacting the plate. ♦ The gripper does not contact the side of the stack. 6 Click Close Gripper. 7 Click Vertical Home. The plate handler raises the arm to its highest position. If the plate contacts the sides of the stack, re-adjust the rotary position of the plate handler arm until the plate moves freely within the stack. Note Contact between the plate and the stack or all stacks may be unavoidable. However, try to minimize the contact as much as possible. 7-36 System Maintenance To re-check the position of input stack 1 (Zymark position 4): Step 8 (continued) Action Click Rotary Offset, and click Yes. The software re-records the rotary position for the input stack 1(Zymark position 4). 9 While holding the plate, click Open Gripper and remove the plate. Defining the Bottom The Automation Controller Software requires a bottom position value for all stacks. of the Stack This value is used to prevent the plate handler arm from colliding or grinding as it moves to the bottom of each stack. To find the bottom of the stack: Step Action 1 Remove all plates from the instrument and the plate handler arm. 2 Place an empty plate into the output stack (Zymark position 0). 3 From the Zymark Twister Software, click position 0. The plate handler arm moves over the output stack. 4 Using the Vertical Positioning commands, lower the plate handler arm until it is just above the stack. 5 Check the rotary position of the plate handler arm to confirm that the gripper: ♦ is centered over the stack ♦ will not contact the sides of the stack when lowered 6 Using the Rotary Adjustment arrows, adjust the rotational position of the gripper so that it is centered over the input stack and will not contact the sides when lowered. 7 Using the Vertical Positioning commands, carefully lower the plate handler arm into the stack. Adjust the Rotary Adjustment value as needed to center the gripper within the stack. 8 Once the gripper is centered within the stack, click Find Plate. The plate handler arm lowers upon the plate. Confirm the following: ♦ The plate is in the middle of the gripper span ♦ The plate sensor switch is contacting the plate ♦ The gripper does not contact the side of the stack 9 Click Close Gripper. The gripper grips the plate between its fingers. 10 Select Vertical Home. The plate handler raises the arm to its highest position. If the plate contacts the sides of the stack, re-adjust the rotary position of the plate handler arm until the plate moves freely in the stack. Note Contact between the plate and the stack or all stacks may be unavoidable. However, try to minimize the contact as much as possible. 11 Using the Vertical Positioning commands, raise and lower plate handler arm several times to check the alignment. 12 Lower the plate handler arm and click Rotary Offset, and click Yes. The software records the rotary position for position 0 (the output stack). System Maintenance 7-37 To find the bottom of the stack: Step (continued) Action 13 Click the Vertical Home. 14 While holding the plate, click Open Gripper and remove the plate. 15 Click the Vertical Adjustment text field, type –3200, and press Enter. The plate handler lowers the arm to a position near the base of the output stack. 16 Carefully lower the plate handler arm until it is approximately 1–2 mm from the bottom of the stack. 17 Click Vertical Offset, click Yes, and record the number in the Vertical Adjustment text field. The software records the vertical position for position 0 (the output stack). 18 Click Vertical Home. The plate handler raises the plate handler arm to its highest position. 19 Click the Vertical Adjustment text field, type the Vertical Offset value determined in step 5, and press Enter. The plate handler lowers the plate handler arm to a Vertical Offset position. 20 If necessary, readjust the Vertical Offset value and repeat steps 6 and 7 until satisfied with the setting. Defining the To define the positions of the remaining stacks: Positions of the Step Action Remaining Stacks 1 Place an empty plate into input stack 2 (Zymark position 5). 2 From the Zymark Twister Software, click position 5. The plate handler arm moves over the input stack. 3 Using the Vertical Positioning commands, lower the plate handler arm until it is approximately 1 cm above the stack and center it using the Rotary Adjustment arrows. 4 Carefully lower the plate handler arm into the stack. Center the gripper as it moves down the stack by adjusting the Rotary Adjustment arrows as needed. 5 Once the plate handler arm is centered within the stack, click Find Plate. The plate handler arm lowers upon the plate. Confirm the following: ♦ The plate is in the middle of the gripper span. ♦ The plate sensor switch is contacting the plate. ♦ The gripper does not contact the side of the stack. 6 Click Close Gripper. 7 Click Vertical Home. The plate handler arm raises to its highest position. If the plate contacts the sides of the stack, re-adjust the rotary position of the plate handler arm until the plate moves freely in the stack. Note Contact between the plate and the stack or all stacks may be unavoidable. However, try to minimize the contact as much as possible. 7-38 System Maintenance To define the positions of the remaining stacks: Step (continued) Action 8 Using the Vertical Positioning commands, raise and lower plate handler arm several times to check the alignment. 9 Click Rotary Offset, and click Yes. The software records the rotary position for the Zymark position 5 (input stack 2). 10 Repeat steps 1–8 for input stacks 3 and 4 to define Rotary Offset values for the remaining positions 6 and 7: Zymark position 7 (input stack 4) Zymark position 6 (input stack 3) 11 Exit the Zymark Twister Software. a. Click Main Menu. The software displays the main menu. b. Click Exit. The software closes. 12 Note A bug in the Zymark Twister Software can cause portions of the program to persist in memory even after the software has been closed. Because the Zymark Twister Software conflicts with the SDS software, these residual elements must be closed within the Windows Task Manager before continuing. Confirm that the Zymark Twister Software has closed by viewing the Task Manager. a. Press the Ctrl + Alt + Del keys in unison. The Windows Security dialog box appears. b. Click Task Manager. The Task Manager dialog box appears. c. Confirm that the Twister software has closed by looking for the Twister software entry in the Task list. If the software is still running, click the software entry and click End Task to exit the software. d. From the File menu, select Exit to quit the Task Manager. Replace the covers for the fixed-position bar code reader and the underlying platform (removed in step 1 on page 7-32). GR2009 13 Fixed-position bar code reader and underlying platform covers System Maintenance 7-39 Aligning the Fixed-Position Bar Code Reader Description The fixed-position bar code reader must be set so that it automatically scans the plate’s bar code as the plate is placed into the instrument tray by the plate handler. Preparing the To move the instrument tray to the Out position: Instrument for the Step Action Alignment Remove the cover for the fixed-position bar code reader. GR2009 1 2 Turn on the computer. 3 Launch the Automation Controller Software. 4 Click Open/Close ( Fixed-position bar code reader cover ). The 7900HT instrument moves the instrument tray to the out position, perpendicular to the instrument. 5 From the File menu, select Exit. The software quits the Automation Controller Software. Positioning the IMPORTANT The instrument tray must be in the OUT position to align the bar code reader. Fixed-Position To position the fixed-position bar code reader: Bar Code Reader Step 1 Action Place a plate with bar code onto the instrument tray. IMPORTANT Orient the plate so that well A1 aligns to the A1 position of the instrument tray and that the bar code faces the fixed-position bar code reader. GR2018 Well position A1 Bar code 2 Select Start > Programs > PSC Laser Data > LDHOST. The LDHOST software launches and displays the LDHOST window. 7-40 System Maintenance To position the fixed-position bar code reader: Step 3 (continued) Action Establish communication with the fixed-position bar code reader as follows: a. Click the Edit button ( ). b. Click the Terminal button ( ). The software opens the Edit Configuration and Terminal dialog boxes. c. From the Device Control dialog box, click the Connect to Device button ( ). The terminal window displays the fixed-position bar code reader response. d. Click OK to close the Information dialog box. The LD Host program communicates with the bar code reader and updates the Edit Configuration dialog box with the current configuration settings. 4 Configure the software for the alignment as follows: a. From the bottom of the Edit Configuration dialog box, locate and click the Op. Modes tab. Op. Modes tab Note You may need to use the arrows located in the bottom of the dialog box to locate the Op. Modes tab. b. From the Operating modes selection group of the Edit Configuration dialog box, click the arrow to the right of the Mode heading and select Test from the drop-down list. Mode drop-down list c. From the Device Control dialog box, click RAM to toggle to EEPROM mode. RAM button d. From the Device Control dialog box, click Send. e. From the Confirm dialog box, click YES to save to EEPROM. The fixed-position bar code reader begins a continuous repeating scan of the bar code. The software updates the Terminal dialog box every 0.5 sec indicating the percentage of accurate reads completed during the 0.5 sec interval. System Maintenance 7-41 To position the fixed-position bar code reader: Step 5 (continued) Action Loosen the black positional adjustment knob on the fixed-position bar code reader, and position the scan head of the reader as far as possible from the plate while maintaining the orientation towards the bar code on the plate (see below) GR2018 Scan head of the fixed-position bar code reader Black positional adjustment knob 6 While watching the Terminal dialog box, slowly adjust the orientation of the fixed-position bar code reader until the percent successful reading displays the highest number possible. Percent successful reads Note It may be helpful to briefly place a sheet of white paper in front of the plate bar code to view the area scanned by the laser. 7 When satisfied with the alignment, tighten the black positional adjustment knob on the fixed-position bar code reader. 8 Restore the fixed-position bar code reader to normal operation: a. From the Edit Configuration dialog box, change from Test back to Serial on Line. Mode drop-down list b. From the Device Control dialog box, confirm that EEPROM is still selected, and click Send. c. From the New Decision dialog box, click YES to save to EEPROM. The bar code reader stops scanning the plate bar code and resumes normal operation. 9 Click Exit ( ) to quit the LDHOST window. The LDHOST window closes. 10 7-42 System Maintenance Replace the cover for the fixed-position bar code reader (from step 1 on page 7-40). Cleaning and Replacing Gripper Finger Pads When to Perform The adhesive used to affix bar code labels to certain brands of microplates can build up on the gripper pads of the Zymark Twister Microplate Handler. Over time, the residue can cause the gripper pads to stick to the microplates while handling them, causing misfeeds. To prevent buildup, inspect the gripper pads monthly and clean or replace the pads as needed. Materials Required The following materials are required to replace the finger pads: Material Part Number Finger Pad Replacement Kit, containing 10 finger pads 4315472 Flat-blade screwdriver, small — Phillips head screwdriver, small — Isopropanol in a squeeze bottle — ! WARNING CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Cleaning the To clean the finger pads, wipe each pad thoroughly with Isopropanol until the residue Finger Pads has been resolved. If the pads appear rough or the adhesive cannot be removed, replace the pads as described below. Replacing the To replace the finger pad(s): Finger Pads Step 1 Action Using the Phillips-head screwdriver, remove the two small Phillips-head screws from the fingers on each side of the gripper, then remove the fingers. Note Move the plate handler arm into any position where it is easy to access the screws. 2 Using a small flat-blade screwdriver, pry the worn finger pads off the fingers. Note The manufacturer recommends replacing all finger pads at the same time. 3 Clean any residual adhesive off the fingers using isopropanol. 4 Remove a replacement finger pad from the paper backing, and place the finger pad on the appropriate finger position. 5 Repeat for the remaining finger pads. 6 Install the fingers with the fingers pointing down and the finger pads toward the center of the gripper. 7 Insert the screws into the fingers and tighten. Note The screws do not automatically align the grippers. Make sure that the finger pads are making good contact with the plate when the arm grips a plate. System Maintenance 7-43 7-44 System Maintenance Section: Maintaining the Computer and SDS Software In This Section This section contains the following information: Topic See Page General Computer Maintenance 7-46 Maintaining the SDS software 7-48 Note The SDS software is a multicomponent system that must be maintained to ensure optimal operation of the ABI PRISM 7900HT Sequence Detection System. Although, most of the maintenance will be completed by an Applied Biosystems service engineer, this section discusses important issues that you should understand. System Maintenance 7-45 General Computer Maintenance Maintenance The computer connected to the 7900HT instrument requires regular maintenance to Schedule ensure reliable operation of the ABI PRISM 7900HT Sequence Detection System components. Applied Biosystems recommends the following tasks as part of routine maintenance of the computer system: Maintenance Task Perform Archive or Remove Old SDS Files Weekly Defragmenting the Hard Drive Monthly or before fragmentation reaches 10% Upgrading the Operating System Software When available/advisable Upgrading the 7900HT SDS Software When available Developing a Data Management Strategy Applied Biosystems recommends developing a strategy for dealing with the files produced by the SDS software. During a single day of real-time operation, the ABI PRISM 7900HT Sequence Detection System can generate over 200 MB of data. Without a strategy for distributing and archiving SDS-related files, the 7900HT instrument can easily fill the hard drive of the computer within just a few weeks of operation. See “Managing Sequence Detection System Data” on page 2-15 for a discussion of management strategies. Archiving SDS Files To conserve space on the computer hard drive, SDS files can be archived using a data compression utility. The compression utility archives files by encoding them in a compressed form, thereby reducing the size of a file. SDS files can be compressed and decompressed many times. Several commercially available compression utilities are available. PKZIP and *.arc are archive formats common to the Microsoft Windows operating system. 7-46 System Maintenance Defragmenting the Applied Biosystems recommends defragmenting the hard drive of the computer Hard Drive attached to the instrument at least once every week or before fragmentation reaches 10%. As the ABI PRISM 7900HT Sequence Detection System is used and files are deleted and created, the free space on the computer hard drive eventually is split into increasingly smaller blocks (called “clusters”). Consequently, as the SDS software creates new files and extends old ones, the computer cannot store each file in a single block. Instead, the system will ‘fragment’ the files by scattering their component pieces across different sectors of the hard drive. The fragmentation of SDS files decreases the performance of both the SDS software and the computer operating system. As the hard drive becomes fragmented, programs take greater time to access files because they must perform multiple seek operations to access the fragments. Several commercially available software utilities are available for repairing fragmented file systems. The software utility defragments broken files by combining their component pieces at a single location on the hard drive, thereby optimizing system performance. Upgrading the Do not upgrade the operating system of the computer connected to the 7900HT Operating System instrument unless instructed to do otherwise by an Applied Biosystems service Software engineer. New versions of the Microsoft Windows operating system can be incompatible with the SDS software and render it and the instrument inoperable. The Applied Biosystems service engineer maintains the operating system software as part of planned maintenance visits. During the visit, the engineer will update the computer operating system as upgrades become available and are validated by Applied Biosystems. System Maintenance 7-47 Maintaining the SDS software Administration IMPORTANT You must have administrator privileges on the computer to install and/or Privileges upgrade the SDS software. Upgrading Applied Biosystems continually develops the SDS software to provide increased the 7900HT functionality and reliability of the ABI PRISM 7900HT Sequence Detection System. As SDS Software updates become available, Applied Biosystems sends notifications of the upgrades to all ABI PRISM 7900HT Sequence Detection System customers. If an upgrade is user-installable, it can be found on the Applied Biosystems company Web site (see Appendix F, “Contacting Technical Support,” to visit the Applied Biosystems Web Site). Note Applied Biosystems service engineers perform regular updates the SDS software during planned maintenance visits. Reinstalling the On rare occasions, when a piece of the SDS software becomes corrupt, it may be Software necessary to re-install the software. In the event that the software must be re-installed, observe the following guidelines to re-install or upgrade the software. 7-48 System Maintenance ♦ Unless instructed to do otherwise, remove the SDS software using the uninstall utility. Do not delete the program folder from the Program Files directory. ♦ Install the SDS software under a user login that has administrator privileges on the computer. ♦ Unless instructed to do otherwise, re-install the SDS software to the same directory as the previous installation. ♦ Review all documentation accompanying the new software (such as installation notes or user bulletin). The updated version of the software may contain new features that require special consideration. Troubleshooting 8 8 In This Chapter This chapter discusses the following topics: Topic See Page Troubleshooting Table 8-2 Low Precision or Irreproducibility 8-4 Background Runs 8-8 Pure Dye Runs 8-10 Real-Time Runs (Quantitative PCR and Dissociation Curves) 8-11 End-Point Runs (Allelic Discrimination) 8-13 Software and 7900HT Instrument 8-14 Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader 8-17 Troubleshooting 8-1 Troubleshooting Table Overview The following table is designed to help you troubleshoot most of the problems you may encounter while using the ABI PRISM® 7900HT Sequence Detection System. The information in the table is arranged by category as follows: ♦ Chemistry problems ♦ Run problems ♦ Instrument and Automation Module Problems Each category contains subcategories, followed by a brief description of the symptoms you might encounter. To use this table, look for the category and the symptom you are experiencing. The page number in the right-hand column corresponds to a description of the possible cause(s) and recommended action(s) for that particular problem. Table 8-1 Troubleshooting Table Category Symptom Page Chemistry and Run Problems Chemistry Low Precision 8-4 Irreproducibility Run Problems Background Runs Software will not extract background data 8-8 Background is too high (greater than 2500) Pure Dye Runs Software will not extract pure dye data 8-10 Raw data from pure dye run appears strange Signals plateau (saturation) Signal is too low (< 10,000 FSU) More than two outliers per dye in a single row 8-2 Troubleshooting Real-Time Runs (Quantitative PCR and Dissociation Curves) 8-11 End-Point Runs (Allelic Discrimination) 8-13 Table 8-1 Troubleshooting Table Category (continued) Symptom Page Instrument and Automation Module Problems Software and 7900HT Instrument SDS software will not launch 8-14 Software crashes/freezes the computer or displays an error message Communication error Thermal cycler errors Automation Controller Software cannot find a plate document file Computer and/or software displays the Run Completed Successfully dialog box but will not respond and appears to be frozen Run will not start Computer is slow when analyzing data, opening or closing dialog boxes, and other software processes. The computer will not logon to the Windows Operating System. The computer will not boot up at all. Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader Plate handler emits grinding noise when picking up or putting down plates 8-17 Plate handler arm contacts racks when retrieving or stacking plates Plate handler arm releases plates awkwardly into the plate stack Reaction plates tip or tilt when placed into the instrument tray by the plate handler arm Plate handler fails to sense or grasp plates Plates stick to the gripper fingers of the plate handler arm Plate handler does not restack plates in original locations Fixed-position bar code reader not reading plate bar codes Troubleshooting 8-3 Low Precision or Irreproducibility Overview There are many reasons why an assay run with the ABI PRISM 7900HT Sequence Detection System can have less than optimal precision. Factors that can affect precision are described in detail below. Factor See Page Improper Threshold Setting 8-4 Imprecise Pipetting 8-5 Non-Optimized Chemistry 8-5 Incomplete Mixing 8-5 Air Bubbles 8-5 Splashing PCR Reagents 8-5 Drops 8-6 Writing on the Reaction Plates 8-6 Fluorescent Contamination on the Plates 8-6 Errors 8-6 Contaminated Sample Block 8-7 Improper or Damaged Plastics 8-7 Low Copy Templates 8-7 Use of Non-Applied Biosystems PCR Reagents 8-7 Improper Threshold The key to high-precision quantitative PCR is accurate detection of the geometric Setting phase. The ABI PRISM 7900HT Sequence Detection System typically delivers sufficient sensitivity so that at least 3 cycles of the geometric phase are visible, assuming reasonably optimized PCR conditions. The SDS software calculates a fixed signal intensity, called a threshold, which each signal generated from PCR amplification must reach before it is recognized as actual amplification. The calculated threshold is an approximation, and should be examined and modified as needed. Modifying the Threshold In a real-time document of the SDS software, the threshold can be modified via the Amplification Plot view following analysis of the run data. See “Setting the Baseline and Threshold Values for the Run” on page 6-10 for more information. 8-4 Troubleshooting Imprecise Pipetting The calculated quantities of target nucleic acid are directly affected by how precisely the template volumes are added to the reaction mixes. Other individually added reagents are also affected by pipetting precision (such as, variable magnesium affects amplification efficiency). Using Master Mixes For this reason, Applied Biosystems highly recommends using a master mix. All common components to a set of reactions should be mixed together and then dispensed to the wells of the plate. Sub-master mixes can be used to further improve the precision of identical replicates. For example, instead of pipetting 5 µL of the same template into four replicate wells, pipette 20 µL of the template into a sub-master mix, then divide the sub-master mix into four equal parts for amplification. When making each master mix, add 5–10% additional volume to compensate for pipetting losses. Using Pipettors Pipetting precision is also improved by: ♦ Calibrating and servicing the pipettors regularly ♦ Pipetting larger volumes ♦ Reducing the number of pipetting steps whenever possible ♦ Increasing the consistency of the pipetting method Consult the manufacturer about the correct method of dispensing liquid volumes accurately from the pipettor. For example, some pipettors are designed to deliver the designated volume at the first plunger stop, so ‘blowing out’ the residue may cause error. Also, before using a new pipettor tip to serially dispense a master mix, wet the tip once by drawing up some of the master mix and dispensing it back into the mix again. Non-Optimized Chemistries that have not been optimized may be susceptible to inconsistencies. To Chemistry maximize precision and reaction efficiency, optimize the primer and probe concentrations of each individual assay used. Refer to the TaqMan Universal PCR Master Mix Protocol (P/N 4304449) for specific information about optimizing probe and primer concentrations for TaqMan-related chemistries. Incomplete Mixing For maximum precision, the PCR master mix must be mixed to uniformity. Once all reaction components are added to master mix, it should be vortexed for 4–5 seconds before aliquotting it to the wells of the plate. Any dilutions performed during the assay should also be vortexed. Air Bubbles Air bubbles in the wells can refract and distort the fluorescent signals. Ideally, the reagents would be applied to the wells using a pipetting technique that does not form air bubbles. However, if a plate does contain air bubbles, they can usually be removed by swinging, tapping, or briefly centrifuging the reaction plate. Splashing PCR If PCR reagents splash the undersides of the optical adhesive covers, the heat from Reagents the lid may bake the liquid to the cover and may distort the signal. If splashing occurs, briefly centrifuge the reaction plate to remove all traces of liquid from the caps. Troubleshooting 8-5 Drops Drops of reagents that cling to the sides of the wells may not contact the thermal cycler sample block and consequently may not amplify. If the drop slides into the mix during PCR, then the amplified products will become diluted and the final result will be less than replicate wells that did not have drops. Therefore, carefully monitor the reaction plate as it is being transferred into the thermal cycler or 7900HT instrument. If you observe any drops, take steps to remove them, such as centrifugation. Writing on the Do not write on any surface of the Optical 384/96-Well Reaction Plates or the Optical Reaction Plates Adhesive Covers. The fluorescent properties of the ink can potentially affect the fluorescence emission from the plate and alter the results. Instead, note the contents of each well on a sheet of paper, or on a printout of the sample setup. Fluorescent Many compounds found in laboratories are fluorescent. If they come in contact with Contamination on certain optical surfaces, such as the optical adhesive covers, the fluorescent results the Plates may be affected. For example, it has been noted that the powder used to lubricate the insides of plastic gloves often contains fluorescent compounds. Use only powder-free gloves and do not needlessly touch the reaction plates or optical adhesive seals. Errors Human errors from time to time are inevitable, such as pipetting into the wrong well, or making a dilution mistake. Human error can be reduced in the following ways: 8-6 Troubleshooting ♦ Perform the assay in a systematic fashion. For example, the pattern of sample positions should be simple (such as avoid putting gaps in the rows). ♦ When pipetting the master mix, look directly down into the reaction plate so that you can verify the transfer of the solution. ♦ If adding a small-volume reagent, such as template, place the drop of liquid on the side of the well. Briefly tap or centrifuge the plate afterwards to bring the droplet down into the well. ♦ After all pipetting is complete, visually inspect all the wells to confirm the presence of the reagent drops. Tapping or centrifuging the reaction plate will cause all the drops to slide down into the wells simultaneously. ♦ When making serial dilutions, be sure to change the pipet tip after each dilution step. ♦ Visually inspect the liquid volumes being pipetted to verify that the volume is approximately correct. A common mistake is using the wrong pipettor volume setting (such as setting 20 µL instead of 2.0 µL). ♦ Visually inspect the volumes of the completed reactions, looking for any wells that have volumes that do not match those of the other wells. Contaminated Any material contaminating the sample block can affect the results. For example, Sample Block mineral oil reduces thermal transfer. Residue from writing on reaction plates darkens the wells, absorbing light. The sample blocks should be periodically inspected for cleanliness. Sample block contamination can be visualized by running a background plate and inspecting the resulting background signal for aberrant peaks above 2500 FSU (see page 7-13). See page 7-11 for instructions on decontaminating the sample block. Improper or Only ABI PRISM optical grade reaction plates, optical adhesive covers, and Damaged Plastics ABI PRISM® optical flat caps should be used with the ABI PRISM 7900HT Sequence Detection System. The plastics that comprise the optical parts undergo special testing for the absence of fluorescent impurities. Optical reaction plates are frosted to improve the degree and precision of light reflection. Bent, creased, or damaged plastics may adversely affect the transmission of fluorescent signal or prevent proper sealing of a well resulting in evaporation, change in sample volume, and altered PCR chemistry. Make sure to use the correct plastics and visually inspect each reaction plate before use. Note See Appendix D, “Kits, Reagents and Consumables,” for a list of compatible consumables and reagents. Low Copy Templates When amplifying samples that contain very low quantities of nucleic acid (generally less than 100 molecules), expect lowered precision due to the Poisson distribution and biochemical effects related to binding probabilities. Low copy templates are also more susceptible to losses due to non-specific adhesion to plastic wells, pipettor tips, etc. The addition of carrier to the sample, such as yeast tRNA or glycogen, can help prevent these losses, increasing the precision and sensitivity of the assay. Use of Non-Applied The Applied Biosystems buffer contains an internal passive reference molecule Biosystems PCR (ROX™), which acts as a normalization factor for fluorescent emissions detected in Reagents the samples (see page A-6). IMPORTANT Non-Applied Biosystems PCR buffers may not contain the ROX passive reference. If running non-Applied Biosystems chemistry, be sure to set the passive reference for your experiment as explained on “Setting the Passive Reference” on page 4-12. Troubleshooting 8-7 Background Runs Background Troubleshooting Table Observation Possible Cause Recommended Action Software will not extract background data During setup, the wrong plate type was assigned to the plate document Run a new background plate document with the proper plate type setting. Background is too high (≥ 2500 FSUa) See below. Sample block contamination a. Construct and run a new background plate. Background is too high (=>2500) Background plate contamination b. See “Isolating Sample Block Contamination” below. a. Fluorescent standard units – The measure of amplitude displayed along the Y-axis of the Background Plot. Isolating Signals exceeding 2500 FSU are considered outside the limit of normal background Sample Block fluorescence and indicates that the either the background plate or the sample block Contamination module may be contaminated. To determine the location of the contamination on the sample block: Step Action 1 If not already open, open the plate document for the background run. 2 From the toolbar, click the Hide/Show System Raw Data Pane button ( ). The SDS software displays the raw data pane for the background run. 3 Select all wells in the plate document. 4 Inspect the raw background data for an aberrant spectral peak or peaks. Wells producing raw spectra that exceed 2500 FSU are considered irregular and could be contaminated. The following figure illustrates the raw data produced by a run on a sample block module containing a contaminated well. Contamination 5 Identify the location(s) of the contaminated well(s) on the sample block by selecting increasingly smaller regions of the plate document (see below). a. The raw data from the selected wells contains the peak. The contaminated well must be in columns 7-12. 8-8 Troubleshooting To determine the location of the contamination on the sample block: Step (continued) Action Example: (continued) b. The raw data from the selected wells does not contain the peak. The contaminated well must be in the last four wells of columns 7-12. c. The raw data from the selected wells contains the peak. The contaminated well must be in the last four wells of columns 10-12. d. The raw data from the selected wells contains the peak. The contaminated well must be in the last two wells of columns 10-12. e. By selecting each of the wells from the last two wells of columns 10-12, the location of the contaminated well (G10) is determined. 6 Repeat step 4 until you identify the location of each contaminated well. 7 Decontaminate the sample block as explained in “Decontaminating the Sample Block” on page 7-11. 8 Run a background plate to confirm that the contaminants have been removed. If the contamination is present after running the background plate for a second time, the background plate is likely to be the source of contamination. Troubleshooting 8-9 Pure Dye Runs Pure Dye Troubleshooting Table Observation Possible Cause Recommended Action Software will not extract pure dye data During plate setup, the wrong plate type was assigned to the plate document Create and run a new pure dye plate document with the proper plate type setting A background plate was not run before the pure dye plate Run a background plate, then run the pure dye plate again Pure dye plate was loaded backwards a. Verify the pure dye wavelengths are as expected. Raw data from pure dye run appears strange (see below) b. Rerun the pure dye plate. Signals plateau (saturation) Intensity is set too high/low Call Applied Biosystems Technical Support. ♦ Evaporation Rerun the pure dye plate. ♦ Contamination If the problem persists, discard the pure dye plate and run a new one. Signal is too low (< 10,000 FSU) More than two outliers per dye in a single row 8-10 Troubleshooting Real-Time Runs (Quantitative PCR and Dissociation Curves) Troubleshooting When faced with irregular data, you can use the SDS software to diagnose some Analyzed Data from chemistry- and instrument-related problems. The following table contains a summary a Real-Time Run of checks for verifying the integrity of your run data and to help you begin troubleshooting potential problems. Troubleshooting Analyzed Real-Time Run Data Analysis View/Description What to look for... Raw Data Plot Signal tightness and uniformity – Do the raw spectra signals from replicate groups and controls exhibit similar spectral ‘profiles’? If not, the plate or sample block could be contaminated. Displays the raw reporter fluorescence signal (not normalized) for the selected wells during each cycle of the real-time PCR. Characteristic signal shape – Do the samples peak at the expected wavelengths? For example, samples containing only FAM™- labeled TaqMan® probes should not produce raw fluorescence in the wavelength of a VIC™ dye component. A signal present in wells that do not contain the dye could indicate that the sample, master mix, or well contains contaminants. Characteristic signal growth – As you drag the bar through the PCR cycles, do you observe growth as expected? Absent growth curves may indicate a pipetting error (well lacks template). Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal. Multicomponent Plot Displays a plot of normalized multicomponent data from a single well of a real-time run. The plot displays the component dye signals that contribute to the composite signal for the well. Correct dyes displayed – Does the plot display all dyes as expected? The presence of an unexpected dye may be the result of an error in detector setup, such as assigning the wrong reporter or quencher dye. ROX fluorescence level – Does the ROX signal fluoresce below the reporter dyes? If not, the lack of reporter fluorescence may be caused by an absence of probe in the well (a pipetting error). Background fluorescence – Do all dyes fluoresce above the background? The Background signal is a measure of ambient fluorescence. If a dye fails to fluoresce above the background, it is a strong indication that the well is missing probes labeled with the dye (well does not contain probe, PCR master mix, or both). MSE Level – The MSE (mean squared error) is a mathematical representation of how accurately the multicomponented data fits the raw data. The higher the MSE value, the greater the deviation between the multicomponented data and the raw data. Troubleshooting 8-11 Troubleshooting Analyzed Real-Time Run Data (continued) Analysis View/Description What to look for... Amplification Plot Correct baseline and threshold settings – Are the baseline and threshold values set correctly? Displays data from real-time runs after signal normalization and Multicomponent analysis. It contains the tools for setting the baseline and threshold cycle (CT) values for the run. Identify the components of the amplification curve and set the baseline so that the amplification curve growth begins at a cycle number that is greater than the highest baseline number. IMPORTANT Do not adjust the default baseline if the amplification curve growth begins after cycle 15. Identify the components of the amplification curve and set the threshold so that it is: ♦ Above the background ♦ Below the plateaued and linear regions ♦ Within in the geometric phase of the amplification curve Irregular amplification – Do all samples appear to have amplified normally? The three phases of the amplification curve should be clearly visible in each signal. Outlying amplification – When the run data is viewed in the CT vs. Well Position plot, do replicate wells amplify comparably? Wells producing CT values that differ significantly from the average for the associated replicate wells may be considered outliers. If a plate produces non-uniformity between replicates, some samples on the plate could have evaporated. Check the seal of the optical adhesive cover for leaks. 8-12 Troubleshooting End-Point Runs (Allelic Discrimination) Troubleshooting When faced with irregular data, you can use the SDS software to diagnose some Analyzed Data from chemistry- and instrument-related problems. The following table contains a summary an End-Point Run of checks for verifying the integrity of your run data and to help you begin troubleshooting potential problems. Troubleshooting Analyzed End-Point Run Data Analysis View/Description What to look for... Raw Data ♦ Signal tightness and uniformity – Do the raw spectra signals from replicate groups and controls exhibit similar spectral ‘profiles’? If not, the plate or sample block could be contaminated. Displays the raw reporter fluorescence signal (not normalized) for the selected wells during each cycle of the PCR. ♦ Characteristic signal shape – Do the samples peak at the expected wavelengths? For example, samples containing only FAM- labeled TaqMan probes should not produce raw fluorescence in the peak wavelength of the VIC dye component. A signal present in wells that do not contain the dye could indicate that the sample, master mix, or well contains contaminants. ♦ Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal. Troubleshooting 8-13 Software and 7900HT Instrument Troubleshooting Troubleshooting Software and Computer Problems Software and Possible Cause Computer Problems Observation SDS software will not launch The software crashes/freezes the computer or displays an error message ♦ Incorrect start-up sequence ♦ Corrupted software Recommended Action Follow the solutions listed until the symptom goes away. 1 ♦ Computer hardware failure a. Turn off the 7900HT instrument. ♦ Operating System (OS) corruption b. Check cable connections. ♦ Loose bar code reader cable c. Restart the computer and logon to the computer. d. Turn on the 7900HT instrument. e. Launch the SDS software. 2 a. Restart the computer and logon to your computer. b. Reinstall the SDS software. c. Launch the SDS software. 3 Contact Applied Biosystems Service for OS problems or if the computer will not boot up at all. You may have to reload the OS from the CDs. 4 Contact Dell for troubleshooting the computer hardware. 8-14 Troubleshooting Communication error Cables are connected incorrectly Check cable connections and COM port setup. See “Instrument Connections” on page 2-11. Thermal cycler errors Sample block module not fully engaged Reseat the sample block module as explained “Replacing the Sample Block” on page 7-4. Automation Controller Software cannot find a plate document file File not in correct location Remove file entry from plate queue and add the file to the plate queue again. Dialog box does not respond to mouse clicks or key strokes Java Runtime Error Click the close box of the dialog box to close it. Troubleshooting Software and Computer Problems (continued) Observation Possible Cause Recommended Action Run will not start No calibration file Perform background and pure dye runs. No background data in calibration file (background run has not been performed) No pure dye data in calibration file (pure dye run has not been performed) See “Performing a Background Run” on page 7-13 and “Performing a Pure Dye Run” on page 7-17. Calibration file does not contain pure dye data for a dye used on the plate document Calibration file was created on another instrument Disk drive containing the plate document has less than 50 MB of free space Check the capacity of the destination drive. If less than 50 MB of free space remains, remove or archive existing data files (see page 7-46). Heated cover cannot reach running temperature because no plate loaded Open the instrument tray and check that the instrument contains a plate. Instrument tray contains a plate Output stack contains a plate or plates Remove all plates from the output stack of the plate handler before starting the queue. Computer is slow when analyzing data, opening or closing dialog boxes, and other software processes Hard drive is fragmented Defragment the hard drive as explained on “Defragmenting the Hard Drive” on page 7-47. Hard drive is almost full Remove or archive existing data files as explained on “Archiving SDS Files” on page 7-46. The computer will not logon to the Windows Operating System Logon window does not appear Restart the computer and logon to your computer. You are not logged on as the Administrator a. Logoff of your computer. After the above solutions have been tried, the problem is still not fixed Contact Dell for troubleshooting the computer hardware or OS. b. Logon again as the Administrator. Troubleshooting 8-15 Troubleshooting Software and Computer Problems (continued) Observation Possible Cause Recommended Action The computer will not boot up at all Cables are not connected or are not seated properly Check the cables. The boot disk is corrupted. a. Boot directly off of the NT Installation CD. b. Boot off of the emergency disk. c. Reload the Windows NT Operating System from the CD. After the above solution has been tried, the problem is still not fixed 8-16 Troubleshooting Contact Dell for troubleshooting the computer hardware. Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader Automation Accessory Troubleshooting Table Observation Possible Cause Recommended Action Plate handler emits grinding noise when picking up or putting down plates Vertical offset too low Plate detector switch set too high Plate handler arm contacts racks when retrieving or stacking plates Plate handler rotary offset is incorrect or vertical offset is too low Re-align the plate handler as explained in “Aligning the Plate Handler” on page 7-32. The plate handler arm releases plates awkwardly into the plate racks Reaction plates tip or tilt when placed into the instrument tray by the plate handler arm Plate handler fails to sense or grasp plates Plate sensor switch not adjusted properly Adjust the plate sensor switch as explained in “Adjusting the Sensitivity of the Plate Sensor Switch” on page 7-28. Gripper pads on the fingers of the plate handler arm are worn or dirty Change the gripper pads as explained in “Cleaning and Replacing Gripper Finger Pads” on page 7-43. Plates stick to the gripper fingers of the plate handler arm Gripper pads are worn or dirty Change the gripper pads as explained in “Cleaning and Replacing Gripper Finger Pads” on page 7-43. Plate handler does not restack plates in original locations Restack when finished Configure the Automation Controller Software to restack the plates as explained in page 4-37. Fixed-position bar code reader not reading plate bar codes Bar code reader is mis-aligned option not selected Bar code reader is broken Re-align the fixed-position bar code reader as explained in “Aligning the Fixed-Position Bar Code Reader” on page 7-40. Troubleshooting 8-17 User Bulletins 9 9 About This Chapter A user bulletin is an advisory issued by Applied Biosystems. User bulletins contain new information, advances, or procedures that may immediately influence your use of Applied Biosystems instruments. This section of the user guide is intended as a storage space for any user bulletins you may receive regarding your ABI PRISM® 7900HT Sequence Detection System. User Bulletins 9-1 Theory of Operation A A In This Appendix This appendix discusses the following topics: Topic See Page Fluorescent-Based Chemistries A-2 Fluorescence Detection and Data Collection A-4 Mathematical Transformations A-5 Real-Time Data Analysis A-7 Theory of Operation A-1 Fluorescent-Based Chemistries Fundamentals of the The PCR reaction exploits the 5´ nuclease activity of AmpliTaq Gold® DNA 5´ Nuclease Assay Polymerase to cleave a TaqMan® probe during PCR. The TaqMan probe contains a reporter dye at the 5´ end of the probe and a quencher dye at the 3´ end of the probe. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. The figure below shows the forklike-structure-dependent, polymerization-associated 5´–3´ nuclease activity of AmpliTaq Gold DNA Polymerase during PCR. Polymerization 5' 3' 5' R Foward Primer Probe Q 3' 5' Strand displacement Reverse Primer R Probe 5' 3' 5' Cleavage R = Reporter Q = Quencher Q 3' 5' 3' 5' R Probe 5' 3' 5' Q 3' 5' 3' 5' Polymerization completed R 5' 3' 5' 3' 5' Probe Q 3' 5' 3' 5' When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Förster-type energy transfer (Förster, 1948; Lakowicz, 1983). During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5´–3´ nucleolytic activity of the AmpliTaq Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3´ end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected. A-2 Theory of Operation Basics of SYBR The SYBR® Green 1 Double-Stranded Binding Dye is used for the fluorescent Green Chemistry detection of double-stranded DNA (dsDNA) generated during PCR. The SYBR Green 1 Dye binds non-specifically to dsDNA and generates an excitation-emission profile similar to that of the FAM™ reporter dye. When used in combination with a passive reference, the SYBR Green 1 Dye can be employed to perform several SDS-related experiments including quantitative PCR and dissociation cure analysis. The following figure illustrates the action of the SYBR Green 1 dye during a single cycle of a PCR. 5´ 3´ 3´ 5´ When added to the reaction, the SYBR Green 1 Dye binds non-specifically to the hybridized dsDNA and fluoresces Dissociation 3´ 5´ 5´ 3´ Denaturation complete, the SYBR Green 1 Dye dissociates from the strand, resulting in decreased fluorescence Polymerization 5´ 3´ Forward Primer 5´ 5´ Reverse Primer 3´ 5´ During the extension phase, the SYBR Green 1 Dye begins binding to the PCR product Polymerization Complete 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ Polymerization is complete and SYBR Green 1 Dye is completely bound, resulting in a net increase in fluorescence Theory of Operation A-3 Fluorescence Detection and Data Collection Fluorescent During PCR, light from an argon ion laser is sequentially directed to each well on the Sequence Detection microplate. The light passes through the ABI PRISM™ Optical Adhesive Cover and the laser excites the fluorescent dyes present in each well of the consumable. The resulting fluorescence emission between 500 nm and 660 nm is collected from each well, with a complete collection of data from all wells approximately once every 7–10 seconds. A system of lenses, filters, and a dichroic mirror focus the fluorescence emission into a grating. The grating separates the light (based on wavelength) into a predictably spaced pattern across a charge-coupled device (CCD) camera. The SDS software collects the fluorescent signals from the CCD camera and applies data analysis algorithms. Charged coupled device array Camera lens Grating Emission filter Beam splitter Laser source Fresnel GR2103 Lens (within Lensplate) 384/96-Well optical plate Side View A-4 Theory of Operation Front View Mathematical Transformations Overview The SDS software performs a series of mathematical transformations on the raw data during an analysis of all end-point and real-time runs. The term raw data refers to the spectral data between 500 nm to 660 nm collected by the SDS software during the sequence detection run. The following section describes the fundamental analysis of raw run data performed on both real-time and end-point run by the SDS software. Multicomponenting Multicomponenting is the term used for distinguishing the contribution each individual dye and background component makes to the fluorescent spectra detected by the 7900HT instrument. During the multicomponent transformation, the SDS software employs several algorithms to separate the composite spectra from the raw spectrum and then to determine the contribution of each dye in the raw data. First, the algorithm eliminates the contribution of background fluorescence in the raw data, by subtracting the background component stored within the background calibration file (see page 7-13). Next, the software employs the extracted pure dye standards (see page 7-17) to express the composite spectrum in terms of the pure dye components. The figure below shows one composite spectrum that represents a fluorescent reading from a single well that contains the passive reference and two fluorogenic probes, labeled with the FAM and VIC™ reporter dyes and a non-fluorescent quencher. The example spectra demonstrate how the overlapping component dye spectra contribute to the composite spectrum. The SDS software multicomponenting algorithm applies matrix calculations to determine the contributions of each component dye spectra. FAM Composite spectrum (FAM + VIC + ROX + Background + MSE) ROX VIC Pure Dye components 500 550 600 Wavelength 650 Background (from background calibration run) Theory of Operation A-5 The software uses the pure dye spectra, generated as part of instrument calibration (see page 7-17), to solve for coefficients a, b, and c in the following equation: Measured spectrum = a ( FAM ) + b ( VIC ) + c ( ROX ) + d ( Background ) + MSE where the coefficients a, b, and c represent each dye component’s contribution to the composite spectrum. Note The example calculation above assumes that pure dye components exist for three dyes (FAM, VIC, and ROX™) and for the instrument background. After solving for a, b, c, and d, the algorithm calculates the mean squared error (MSE), which measures how closely the collective multicomponent spectrum conforms to the raw spectra. The figure below shows a typical display of the contribution of each component spectra for one well. Multicomponent Plot Temperature Fluorescence FAM component 12000 8000 VIC component 4000 ROX component 0 93 Background component 83 MSE 73 63 53 0:10 0:30 0:50 6978 1:10 Time 1:30 Normalization of While multicomponenting illustrates absolute change in emission intensity, the SDS Reporter Signals software displays cycle-by-cycle changes in normalized reporter signal (Rn). The SDS software normalizes each reporter signal by dividing it by the fluorescent signal of the passive reference dye. Because the passive reference is a component of the PCR master mix, it is present at the same concentration in all wells of the plate. By normalizing the data using the passive reference, the software can account for minor variations in signal strength caused by pipetting inaccuracies and make better well-to-well comparisons of reporter dye signal. Note For the example above, the resulting data from the normalizing is displayed as FAM Rn and VIC Rn. A-6 Theory of Operation Real-Time Data Analysis Kinetic Analysis/ The 7900HT instrument can be used to determine the absolute or relative quantity of a Quantitative PCR target nucleic acid sequence in a test sample by analyzing the cycle-to-cycle change in fluorescence signal as a result of amplification during a PCR. This form of quantitative PCR analysis, called “kinetic analysis,” was first described using a non-sequence-specific fluorescent dye, ethidium bromide, to detect PCR product (Higuchi et al., 1992; Higuchi et al., 1993). The use of TaqMan probes and reagents further enhances the method by providing sequence-specific amplification of multiple targets for ‘comparative’ or ‘relative’ quantification. The fewer cycles it takes to reach a detectable level of fluorescence, the greater the initial copy number of the target nucleic acid. Amplification Plot 8.0 Phase 3, plateau 2.0 1.0 Phase 2, linear Rn Phase 1, geometric 0.0 0 10 20 30 40 Cycle Number When graphed in real-time on a linear scale, normal amplification of PCR product generates a curve similar to the one shown in the figure above. This ‘amplification’ curve consists of three distinct regions that characterize the progression of the PCR. Phase 1: Geometric (Exponential) Detection of the high-precision geometric phase is the key to high-precision quantitative PCR. The geometric phase is a cycle range of high precision during which is characterized by a high and constant amplification efficiency. It occurs between the first detectable rise in fluorescence and before the beginning of the Linear phase. When plotted on a log scale of DNA vs. cycle number, the curve generated by the geometric phase should approximate a straight line with a slope. The 7900HT instrument typically delivers sufficient sensitivity to detect at least 3 cycles in the geometric phase, assuming reasonably optimized PCR conditions. Theory of Operation A-7 Phase 2: Linear The linear phase is characterized by a leveling effect where the slope of the amplification curve decreases steadily. At this point, one or more components have fallen below a critical concentration and the amplification efficiency has begun to decrease. This phase is termed linear, because amplification approximates an arithmetic progression, rather than a geometric increase. Because the amplification efficiency is continually decreasing during the Linear phase, it exhibits low precision. Phase 3: Plateau Finally, the amplification curve achieves the plateau phase at which time the PCR stops and the Rn signal remains relatively constant. Determining Initial Template Concentration and Cycle Number At any given cycle within the geometric phase of PCR, the amount of product is proportional to the initial number of template copies. When one template is diluted several times, as with the RNase P target in the TaqMan® RNase P Instrument Verification Plate (see Appendix D), the ratio of template concentration to detectable signal is preserved within the exponential phase for all dilutions (see below). This relationship appears to change as rate of amplification approaches a plateau. 8.0 2.0 1.0 Rn 20000 10000 5000 2500 1250 0.0 0 10 20 Cycle Number A-8 Theory of Operation 30 40 Fluorescence vs. When using TaqMan fluorogenic probes with the 7900HT instrument, fluorescence Amplified Product emission increases in direct proportion to the amount of specific amplified product. As the figure on page A-8 demonstrates, the graph of normalized reporter (Rn) vs. cycle number during PCR appears to have three stages. Initially, Rn appears as a flat line because the fluorescent signal is below the detection limit of the Sequence Detector. In the second stage, the signal can be detected as it continues to increase in direct proportion to the increase in the products of PCR. As PCR product continues to increase, the ratio of AmpliTaq Gold polymerase to PCR product decreases. When template concentration reaches 10-8 M, PCR product ceases to grow exponentially. This signals the third stage of Rn change, which is roughly linear and finally reaches a plateau at about 10-7 M (Martens and Naes, 1989). The progressive cleavage of TaqMan fluorescent probes during the PCR makes possible the correlation between initial template concentration and the rise in fluorescence. As the concentration of amplified product increases in a sample, so does the Rn value. During the exponential growth stage (the geometric phase), the relationship of amplified PCR product to initial template can be shown in the following equation: Nc = N( 1 + E )c where Nc is the concentration of amplified product at any cycle, N is the initial concentration of target template, E is the efficiency of the system, and c is the cycle number. Theory of Operation A-9 Calculating The ABI PRISM 7900HT Sequence Detection System creates quantifiable Threshold Cycles relationships between test samples based on the number of cycles elapsed before achieving detectable levels of fluorescence. Test samples containing a greater initial template number cross the detection threshold at a lower cycle than samples containing lower initial template. The SDS software uses a Threshold setting to define the level of detectable fluorescence. The threshold cycle (CT) for a given amplification curve occurs at the point that the fluorescent signal grows beyond the value of the threshold setting. The CT represents a detection threshold for the 7900HT instrument and is dependent on two factors: ♦ Starting template copy number ♦ Efficiency of DNA amplification the PCR system How the SDS Software Determines CTs To determine the CT for an amplification plot, the SDS software uses data collected data from a predefined range of PCR cycles called the ‘baseline’ (the default baseline occurs between cycles 3 and 15). First, the software calculates a mathematical trend based on the baseline cycles’ Rn values to generate a baseline subtracted amplification plot of ∆Rn versus cycle number. Next, an algorithm searches for the point on the amplification plot at which the ∆Rn value crosses the threshold setting (the default threshold setting is 0.2). The fractional cycle at which the intersection occurs is defined as the threshold cycle (CT) for the plot. Note It may be necessary to adjust the baseline and threshold settings to obtain accurate and precise data. For further information on resetting the baseline and threshold settings, see “Setting the Baseline and Threshold Values for the Run” on page 6-10. 8.0 2.0 1.0 1250 Rn Threshold setting 0.0 0 10 20 Cycle Number A-10 Theory of Operation 30 40 Threshold cycle (CT) for the 1250 copy well Significance of Beginning with the equation describing the exponential amplification of the PCR: Threshold Cycles X n = Xm ( 1 + EX ) n–m where: Xn = number of target molecules at cycle n (so that n ≥ m) Xm = number of target molecules at cycle m (so that m ″ n) EX = efficiency of target amplification (between 0–1) n-m = number of cycles elapsed between cycle m and cycle n Amplicons designed and optimized according to Applied Biosystems guidelines (amplicon size <150 bp) have amplification efficiencies that approach 100 percent. Therefore EX=1 so that: X n = Xm ( 1 + 1 ) = Xm ( 2 ) n–m n–m To define the significance in amplified product of one thermal cycle, set n - m = 1 so that: X 1 = X0 ( 2 ) 1 = 2X 0 Therefore, each cycle in the PCR reaction corresponds to a two-fold increase in product. Likewise, a change in threshold cycle number of one must equate to a two-fold difference in initial template concentration. Theory of Operation A-11 Importing and Exporting Plate Document Data B B In This Appendix This appendix discusses the following topics: Topic Importing Plate Document Setup Table Files See Page B-2 Setup Table File Format B-4 Exporting Graphics B-8 Exporting Plate Document Data B-9 Importing and Exporting Plate Document Data B-1 Importing Plate Document Setup Table Files About the Import The SDS software features the ability to import setup table information (detector, Function detector task, marker, and sample name layouts) into a plate document from a tab-delimited text file. The import feature is designed to be a time-saving device that facilitates the exchange of setup information between other programs and the SDS software. Instead of setting up plate documents individually, a third-party program can be used to construct setup table files which can then be imported into plate documents for use. To guarantee a successful incorporation of setup information from a text file to the plate document, the file must: ♦ Be saved in a tab-delimited text format ♦ Conform to the setup table file formats described on page B-4 Creating and Importation of setup table data into a plate document is accomplished in three major Importing Setup steps. Table Data into a Plate Document Creating an Empty Setup Table File The first step in the procedure is to export a setup table file from a blank plate document. Note The blank setup table file can be created using a secondary application (such as Microsoft Excel or a text editor) so long as it is saved in tab-delimited format and is configured according to the file structure explained on page B-4. To export a blank setup table file using the SDS software: Step 1 Action Launch the SDS software. 2 From the File menu, select New. 3 Configure the New Document dialog box with the correct assay type and plate format for your experiment, and click OK. 4 From the File menu, select Export. 5 From the Look In text field of the Export dialog box, navigate to the directory you would like to receive the exported file. 6 From the Export drop-down list, select Setup Table. 7 Select the All Wells radio button. 8 Click the File name text box, and type a name for the file. 9 Click Export. The software exports the setup table data for the empty plate document as a tab-delimited text file. 10 Configure the setup table file with plate document information (detector, task, marker, and sample data) as explained on page B-3. B-2 Importing and Exporting Plate Document Data Configuring the Setup Table File with Plate Document Information The second step in the procedure is to import the setup table file into a secondary application, configure it with sample and detector information, and then save the completed setup table file in tab-delimited format. To configure the setup table file with information: Step Action 1 Launch the application that you want to use to edit the setup table file. 2 Import the setup table file from the previous procedure as tab-delimited text. If using a spreadsheet application to edit the setup table file, the application automatically parses the tab-delimited information into the cells of a spreadsheet. 3 Configure the setup table file with sample and detector information according to the file structure explained on page B-5. 4 Save the setup table file in tab-delimited format. 5 Import the completed setup table file into an empty plate document as explained below. Importing the Completed Setup Table File into a Plate Document The final step in the procedure is to import the completed setup table tab-delimited file into an empty plate document. To import setup information from a tab-delimited text file to a plate document: Step 1 Action If the plate document created in “Creating an Empty Setup Table File” on page B-2 is still open in the SDS software, continue to step 3. Otherwise, create a plate document to receive the setup table data as follows: a. Launch the SDS software. b. Create or open a plate document to receive the information from the text file. 2 Choose one of the following options: ♦ From the toolbar, click the Import button ( ). ♦ From the File menu, select Import. 3 From the Look In text field of the Import dialog box, navigate to and select the completed tab-delimited setup table file from step 4 in the previous procedure. 4 Click Import. The software imports the setup table information from the text file and automatically configures the plate document plate grid and setup table with detector, detector task, marker, and sample data. Importing and Exporting Plate Document Data B-3 Setup Table File Format Example Setup To guarantee a successful importation of setup table data into a plate document, the Table Files imported setup table file must be configured in the correct format for the assay type. The following figures illustrate the orientation of information in tab-delimited setup table files as viewed in a Microsoft® Excel spreadsheet document. The numbered elements of the setup table files are explained on page B-5. Example Setup Table File from an Allelic Discrimination Run 1 2 3 4 5 6 7 8 Example Setup Table File from an Absolute Quantification Run 1 2 3 4 5 6 7 8 B-4 Importing and Exporting Plate Document Data About the Setup This section explains the elements of setup table files shown on the previous page. Table File Format The following table describes the conventions used in the rest of this section. Format/Symbol Definition Text appearing in bolded courier font must be applied to a setup table file exactly as appears in this document. courier Text appearing in italic courier font must be substituted with custom values when applied to a setup table file. italic [ required text ] Text appearing between brackets is required information in setup table files. All information within the brackets must be present in the setup table file for the SDS software to import it. { required text } Text appearing between braces is optional in setup table files. The tab character (the equivalent of pressing the Tab key) <tab> The carriage-return character (the equivalent of pressing the <cr> Enter key) IMPORTANT To guarantee a successful importation of the setup table file into a plate document, the file can must contain all of the sections in the following table in the order that they appear in this document. Setup Table Elements Number 1 Contents Description File Version This line defines the version of SDS Assay Plate File format used to generate the document. Format: [ *** SDS Setup File Version <tab> version number <cr> ] Example: *** SDS Setup File Version 2 2 Plate Size This line defines the number of wells in the plate modeled by the file (384 or 96). Format: [ *** Output Plate Size <tab> number of wells <cr> ] Example: *** Output Plate Size 3 Plate ID 384 This line defines the ID of the Assay Plate. Normally this will be a bar code that is printed on the plate. Format: [ *** Output Plate ID <tab> plate id <cr> ] Example: *** Output Plate ID 384N75822034 Importing and Exporting Plate Document Data B-5 Setup Table Elements Number (continued) Contents Detector Definitions 4 Number of Detectors Description Element numbers 4 to 6 define the detectors that will appear in the well descriptions that follow in a later section. The detector definition consists of three sections: the declaration of the number of detectors, the detector list header, and the detector list. This line defines the total number of detectors on the plate. Format: [ *** Number of Detectors <tab> number of detectors <cr> ] Example: *** Number of Detectors 5 Detectors List Header 5 This line contains the column headings for the Detector Definitions section of the setup table file that make the file easier to edit using a program such as Microsoft Excel. Format: [ Detector <tab> Reporter <tab> Quencher <tab> Description <tab> Comments <tab> Sequence <cr> ] Example: Detector 6 Detectors List ReporterQuencher Description Comments Sequence The detector list consists of one or more lines displaying the information for each detector used on the plate document. ♦ The Detectors List section must contain one line (or definition) for each detector present on the plate. ♦ The number of lines in the Detectors List section must be equal to the number defined in the Number of Detectors section (see number 4 above). ♦ Leave blank the Quencher Dye entry for detectors created for the SYBR Green I Dye or probes labeled with a non-fluorescent quencher. ♦ The Sequence text value contains the name of the Allelic Discrimination marker associated with the detector. Because markers are used exclusively in allelic discrimination plate documents, assign Sequence text values to detector definitions of allelic discrimination setup table files only. Format for a single detector: [ detector name <tab> reporter dye <tab> quencher dye <tab> description <tab> comments <tab> sequence <cr> ] Example for an allelic discrimination setup table file: CYP 2C9*2.1 FAM PDAR CYP 2C9*2 Allele 1Example ProbeCYP 2C9*2 CYP 2C9*2.2 VIC PDAR CYP 2C9*2 Allele 2Example ProbeCYP 2C9*2 Example for an absolute quantification setup table file: GAPDH VIC GAPDH Probe Example Probe SYBR Green SYBR SYBR Green I Example Probe RNase P FAM RNase P Probe Example Probe TAMRA B-6 Importing and Exporting Plate Document Data Setup Table Elements Number Contents Assay Plate Wells 7 (continued) Description Element numbers 7 and 8 define the contents of the wells on the plate. The Assay Plate Wells definition consists of two sections: the Well List Header and the Well Definition List. Well List Header This line contains the column headings for the Assay Plate Wells section of the setup table file that make the file easier to edit using a program such as Microsoft Excel. Format: [ Well <tab> Sample Name <tab> Detector <tab> Task <tab> Quantity ] { ... <tab> Detector <tab> Task <tab> Quantity } [ <cr> ] Example: Well 8 Sample Name Well Definition List Detector Task Quantity ...Detector Task Quantity This section defines the contents of the plate wells. The setup table file must contain a definition for each well used on the plate. Each well definition list consists of one string of characters terminated by a <cr>. The definition can be of three main functional divisions: ♦ Well number – The first tab-delimited text block defines the number of the well on the plate. Well numbers start at 1 for well A-1 (upper-left corner of the plate) and increases from left to right and from top to bottom. The wells must be listed in order (1,2,3,…). ♦ Sample name – The second tab-delimited text block defines the name of the sample assigned to the well. ♦ Detector assignments – The remaining tab-delimited text blocks for the well definition define the detectors assigned to the well. Each detector is represented by three text blocks that define the following information: – The name of the detector – The task assignment of the detector for the well (UNKN - Unknown, STND - Standard, NTC - No Template Control) – The quantity assignment of the detector for the well (For wells containing standards, assign the quantity for the standard sample in initial copy number. For all other wells, assign the quantity value as 0.) To assign more than one detector to a well, then repeat the detector definition text blocks for each detector. There is no limit to the number of detectors that can appear in a well. IMPORTANT All detectors that appear in this section must have been previously defined in the Detector Definitions section (elements 4–6). Format for a single Well: [ Well number <tab> SDS Sample Name <tab> Detector name <tab> Detector task <tab> Detector quantity ] { <tab> Detector name <tab> Detector task <tab> Detector quantity … <tab> Detector name <tab> Detector task <tab> Detector quantity } [ <cr> ] Example for allelic discrimination setup table files: 1 2 3 Sample 1 Sample 2 ... CYP 2C9*2.1 CYP 2C9*2.1 UNKN UNKN 0 0 CYP 2C9*2.2 UNKN CYP 2C9*2.2 UNKN 0 0 Example for absolute quantification setup table files: 1 2 3 4 5 Sample Sample Sample Sample Sample 1 2 3 4 5 GAPDH GAPDH GAPDH GAPDH GAPDH UNKN UNKN STND STND NTC 0 0 20000 15000 0 Importing and Exporting Plate Document Data B-7 Exporting Graphics Exporting a Plot as a The SDS software can export most panes and plots of the plate document as JPEG JPEG Graphic File (Joint Photographic Experts Group) graphic files. The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software. To export an element of a plate document as a graphic: Step Action 1 Click the plot or grid you want to export. 2 Choose from the following: ♦ If exporting a plot, adjust its dimensions (length and width) as you want them to appear in the exported graphic file. The exported graphic file retains the dimensions of the original screen element. ♦ If exporting the plate grid, do not adjust the size of the wells. The software captures the whole grid regardless of the size of the view. 3 Right-click the plot or grid, and select Save Plot/Grid to Image File from the contextual menu. Note If a pane cannot be exported as a graphic, the contextual menu will not contain the Save Plot/Grid to Image File option. 4 From the Save As dialog box, navigate to the directory you want to receive the exported graphic file. 5 Click the File name text field, and type a name for the new file. 6 Click Save. The software saves the plot or grid as a JPEG graphic in the designated directory. B-8 Importing and Exporting Plate Document Data Exporting Plate Document Data Exporting Data from The SDS software can export raw or analyzed data in tab-delimited (*.txt) format for all a Plate Document or a select group of wells on a plate document. The exported files are compatible with most spreadsheet applications. To export an element of a plate document: Step 1 Action Click the plate document to select it. Note The plate document must be the top-most object in the workspace. 2 From the File menu, select Export. 3 Navigate to the directory you would like to receive the exported file(s). 4 Click the Export drop-down list, and select the type of data you would like the software to export. Data Type The exported file contains… Background Spectra Fluorescence readings for each well from the background component used to analyze the run Clipped Average Rn and ∆Rn of the last three data points collected during the extension phase of each cycle repetition for each well Dissociation Curve ♦ Temperature Data – For each well in use on the plate, the file displays the calculated temperature of the wells during each data collection reading of the temperature ramp. ♦ Raw Data – For each well in use on the plate, the file displays the Rn of the well during each data collection reading of the temperature ramp. ♦ Derivative Data – For each well in use on the plate, the file displays the first derivative data during each data collection reading of the temperature ramp. Multicomponent ♦ Calculated amounts the dye components in a single well throughout all stages of the PCR that were labeled with data collection icons ♦ Pure spectra component data ♦ Calculated inverse matrix ♦ Singular values of the inverse matrix Pure Spectra Fluorescence readings for each well from the pure spectra calibration component used to analyze the run Raw Spectra Unmodified fluorescence readings taken for each spectral bin during the course of the run When exported, the software creates a directory and saves each the raw spectra data for each well in a separate *.txt file. Results Table The contents of the results table of an analyzed plate document Note The contents of the exported data varies depending on the type of plate document. Setup Table The contents of the results table of a plate document prior to analysis The contents of the exported data varies depending on the type of plate document used to produce it. See page B-4 for a detailed description of the Setup Table file. Importing and Exporting Plate Document Data B-9 To export an element of a plate document: Step 5 (continued) Action Select the appropriate well radio button as follows: To export data from… Select the… all wells on the plate document All Wells radio button selected wells of the plate grid only Selected Wells radio button 6 From the files of type drop-down list, select the appropriate format for the exported data. 7 Click the File name text box, and type a name for the exported file. 8 Click Export. The software saves the exported data to the designated location. B-10 Importing and Exporting Plate Document Data Designing TaqMan Assays C C In This Appendix This appendix discusses the following topics: Topic Assay Development Guidelines See Page C-2 Design Tips for Allelic Discrimination Assays C-5 Design Tips for Quantitative PCR Assays C-6 Designing TaqMan Assays C-1 Assay Development Guidelines TaqMan Assay To develop custom TaqMan 5´ nuclease assays: Development Step Action Program See Page 1 Identify target sequence(s). C-2 2 Design the TaqMan® probes and the forward and reverse primers. C-3 3 Order reagents. C-4 4 Quantitate the concentrations of the probes and primers. C-4 5 Prepare the master mix. C-4 6 Optimize the primer concentrations. C-4 7 Run the assay. C-4 Identify Target A target template is a DNA, cDNA, RNA, or plasmid containing the nucleotide Sequence(s) sequence of interest. For optimal results, the target template should meet the following requirements: ♦ The target nucleotide sequence must contain binding sites for both primers (forward and reverse) and the fluorogenic probe. ♦ Short amplicons work best. Amplicons ranging from 50–150 bp typically yield the most consistent results. ♦ If designing assays for quantitative PCR, see “Design Tips for Quantitative PCR Assays” on page C-6 for additional recommendations. C-2 Designing TaqMan Assays Design Probes and The following sections contain general guidelines for designing primers and probes. Primers For specific design tips, refer to the appropriate section: for Allelic Discrimination see page C-5 and for Quantitative PCR see page C-6. Design Probe(s) for the Assay Adhere to the following guidelines when designing TaqMan probes: ♦ Keep the G-C content in the range of 30–80%. ♦ Avoid runs of an identical nucleotide (especially guanine, where runs of four or more Gs should be avoided). ♦ No G on 5´ end. ♦ Keep the melting temperature (Tm) in the range of 68-70 °C for quantitative PCR and 65-67 °C for allelic discrimination (using the Primer Express™ software). ♦ Select the strand that gives the probe with more Cs than Gs. ♦ For allelic discrimination (see page C-5): ♦ – Adjust probe length so that both probes have the same Tm. – Position the polymorphism site approximately in the center of each probe. For multiplex PCR applications (involving multiple probes), design the probes with different fluorescent reporter dyes as explained below: Reporter Dyea Application Allelic Discrimination First Probe Second Probe FAM™ VIC™ a. The use of the FAM and VIC reporter dyes for multiplex applications provides the greatest degree of spectral separation. Design Primers for the Assay Adhere to the following guidelines when designing primers for 5´-nuclease assays: ♦ Keep the G-C content in the range of 30–80%. ♦ Avoid runs of an identical nucleotide (especially guanine, where runs of four or more bases should be avoided). ♦ Keep the Tm in the range of 58-60 °C (using the Primer Express™ software). ♦ Limit the G and/or C bases on the 3´ end. The five nucleotides at the 3´ end should have no more than two G and/or C bases. ♦ Place the forward and reverse primers as close as possible to the probe without overlapping the it. ♦ Use an annealing temperature of 60 °C for quantitative PCR, and 62 °C for allelic discrimination (except for TaqMan® PDARs for Allelic Discrimination). Designing TaqMan Assays C-3 Order Reagents Note Because part numbers can change as new and improved products are introduced. Contact your Applied Biosystems sales representative for specific ordering information. You will need the following reagents and equipment to create your own applications: ♦ Custom Synthesized TaqMan Probes ♦ Sequence Detection Primers ♦ TaqMan® Universal PCR Master Mix (optimized for TaqMan reactions containing AmpliTaq® Gold DNA Polymerase, AmpErase® UNG, dNTPs with dUTP, ROX™ Passive Reference I, and optimized buffer components) IMPORTANT PCR master mix used with the 7900HT instrument must contain a passive reference dye. The SDS software uses the signal from the passive reference to normalize the reporter fluorescence making well-to-well comparisons possible. All Applied Biosystems master mix products contain an optimal concentration of the Passive Reference I. ♦ ABI Prism® Optical Reaction Plates ♦ ABI Prism® Optical Adhesive Covers or ABI PRISM® Optical Flat Cap Strips ♦ TaqMan Spectral Calibration Reagents ♦ Centrifuge with 384- or 96-well plate adapter ♦ Deionized water or Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) ♦ Disposable Gloves Quantitate the Use a spectrophotometric method to determine the concentrations of the probes and Probes and Primers primers received. See the TaqMan Universal PCR Master Mix Protocol (P/N 4304449) for specific information about primer and probe quantification. Prepare Master Mix Refer to the TaqMan Universal PCR Master Mix Protocol (P/N 4304449) for specific information about preparing the master mix for use. IMPORTANT PCR master mix used with the 7900HT instrument must contain a passive reference dye. The SDS software uses the signal from the passive reference to normalize the reporter fluorescence making well-to-well comparisons possible. All Applied Biosystems master mix products contain an optimal concentration of the ROX passive reference dye. Note Applied Biosystems protocols are available on the Applied Biosystems Company Web Site, see Appendix F, “Contacting Technical Support,” for more information. Optimize Refer to the TaqMan Universal PCR Master Mix Protocol (P/N 4304449) for specific Primer/Probe information about preparing the master mix for use. Concentrations Note Applied Biosystems protocols are available on the Applied Biosystems Company Web Site, see Appendix F, “Contacting Technical Support,” for more information. Run Your Custom Run your experiment. Assay Note If conducting a quantitative PCR experiment, consider the use of replicate assays to enhance the precision of you data. C-4 Designing TaqMan Assays Design Tips for Allelic Discrimination Assays Discrimination by By using different reporter dyes, cleavage of multiple probes can be detected in a Multiple Probes single PCR. One application of this multi-probe capability is to use allele-specific probes to distinguish genetic polymorphisms (Bloch, 1991, Lee et al., 1993). Probes that differ by as little as a single nucleotide will exhibit allele-specific cleavage. This is true even for probes with a reporter on the 5' end and the non-fluorescent quencher on the 3´ end (Bloch, 1991). TaqMan Probe IMPORTANT When designing probes, it is important to consider probes from both strands. Design Guidelines Follow the guidelines in the table below for designing TaqMan MGB probes: Priority 1 Guideline Avoid probes with a guanine residue at the 5´ end of the probe. A guanine residue adjacent to the reporter dye will quench the reporter fluorescence, even after cleavage. 2 Select probes with a Primer Express software–estimated Tm of 65–67 °C. 3 Make the TaqMan MGB probes as short as possible, but no fewer than 13 nucleotides in length. 4 Avoid runs of an identical nucleotide. This is especially true for guanine, where runs of four or more should be avoided. 5 Position the polymorphic site in the central third of the probe. Note The polymorphic site can be shifted toward the 3´ end to meet the above guidelines, however, the site must be located more than two nucleotides upstream from the 3´ terminus. The following figure illustrates the placement of a polymorphism in an example probe (N = Nucleotide). First, try to position the polymorphic site in the central third of the probe. Do not place it here. 5´ 3´ N N N N N N N N N N N N N N N N N N N N N Polymorphism If necessary, place the polymorphism here. Designing TaqMan Assays C-5 Design Tips for Quantitative PCR Assays Selecting an Amplicon Site for Gene Expression Assays Selecting a good amplicon site ensures amplification of the target mRNA without co-amplifying the genomic sequence, pseudogenes, and related genes. Applied Biosystems recommends the following guidelines when selecting an amplicon site for quantification assays: ♦ Primers and probes must be designed following the “Assay Development Guidelines” on page C-2. ♦ The amplicon should span one or more introns to avoid amplification of the target gene in genomic DNA. ♦ The primer pair has to be specific to the target gene and does not amplify pseudogenes or other related genes. ♦ Test amplicons and select those that have the highest signal-to-noise ratio (such as those yielding low CTs with cDNA and no amplification with no template control or genomic DNA). ♦ If no good sequence is found, it may be necessary to examine the sequence and redesign the amplicon or simply screen for more sites. If the gene you are studying does not have introns, then you cannot design an amplicon that will amplify the mRNA sequence without amplifying the genomic sequence. In this case, it may be necessary to run RT minus controls. Selecting and Preparing Standards for Absolute Quantification To ensure accurate results, the standards used for absolute quantification must be carefully engineered, validated, and quantified before use. Consider the following critical points for the proper use of absolute standard curves: ♦ The DNA or RNA used must be a single, pure species. For example, plasmid DNA prepared from E. coli often is contaminated with RNA, which increases the A260 measurement and inflates the copy number determined for the plasmid. ♦ In general, DNA cannot be used as a standard for absolute quantification of RNA because there is no control for the efficiency of the reverse transcription step. ♦ Absolute quantities of the standard must be known by some independent means. Plasmid DNA or in vitro transcribed RNA are commonly used to prepare absolute standards. Concentration is measured by A260 and converted to the number of copies using the molecular weight of the DNA or RNA. ♦ Consider the stability of the diluted standards, especially for RNA. Divide diluted standards into small aliquots, store at -80 °C, and thaw only once before use. An example of the effort required to generate trustworthy standards is provided by Collins et al. (Anal. Biochem. 226:120-129, 1995), who reported on the steps they used in developing an absolute RNA standard for viral quantification. ♦ Pipetting must be accurate because the standards must be diluted over several orders of magnitude. Plasmid DNA or in vitro transcribed RNA must be concentrated in order to measure an accurate A260 value. The concentrated DNA or RNA must then be diluted 106 –1012 -fold to be at a concentration similar to the target in biological samples. C-6 Designing TaqMan Assays Kits, Reagents and Consumables D D In This Appendix This appendix discusses the following topics: Topic Interchangeable Sample Block Modules and Accessories See Page D-2 Consumables and Disposables D-3 Instrument Maintenance and Verification D-4 TaqMan Pre-Developed Assays and Reagents D-5 Custom Oligonucleotide Synthesis D-5 Note Part numbers listed within this appendix are for customers within the United States. Contact your Regional Sales Office for local Part numbers and prices (see Appendix F, “Contacting Technical Support,” for a list of telephone and Fax numbers). Kits, Reagents and Consumables D-1 Interchangeable Sample Block Modules and Accessories The 7900HT instrument features a Peltier-based, interchangeable sample block module based on the technology established in the GeneAmp ® PCR System 9700 thermal cycler. The use of an interchangeable sample block module: ♦ Reduces instrument downtime by allowing immediate replacement of the block. ♦ Permits easy access to the sample block for troubleshooting and maintenance (see page 7-11). ♦ Supports multiple consumable formats. ♦ Provides several different modes of operation (including Max mode and programmable temperature ramps). Part No. a Description Quantity 384-Well Interchangeable Sample Block Module for the ABI PRISM® 7900HT Sequence Detection System 1 kit Includes a 384-Well Sample Block Module, a 384-well plate adapter, and a Sequence Detection Systems 384-Well Spectral Calibration Kit (P/N 4323977) a 96-Well Interchangeable Sample Block Module for the ABI PRISM® 7900HT Sequence Detection System Includes a 96-Well Sample Block Module, a 96-well plate adapter, and an ABI PRISM™ 7900HT Sequence Detection Systems 96-Well Spectral Calibration Kit (P/N 4328639) a. contact your local Applied Biosystems Sales and Service Office for information. D-2 Kits, Reagents and Consumables 1 kit Consumables and The ABI PRISM 7900HT Sequence Detection System can run both: Disposables ♦ ABI PRISM™ Optical 384-Well Reaction Plates sealed with ABI PRISM™ Optical Adhesive Covers ♦ ABI PRISM™ 96-Well Reaction Plates sealed with ABI PRISM Optical Adhesive Covers or ABI PRISM® Optical Caps (flat cap strips only) IMPORTANT Do not use MicroAmp® Optical Caps or MicroAmp® Optical Tubes with the 7900HT instrument. The instrument is not designed to run MicroAmp consumables which may damage its internal components if used. Note ABI PRISM Optical Reaction Plates are designed specifically for fluorescence-based PCR chemistries and are frosted to minimize external fluorescent contamination. Before running prepared ABI PRISM Optical Reaction Plates on the 7900HT instrument, each plate must be sealed with an ABI PRISM Optical Adhesive Cover. Applied Biosystems’ optical adhesive covers are specifically designed to permit the transmission of light to and from the wells of the optical plate. Part No. Description Quantity ABI PRISM™ Optical Adhesive Covers 4313663 ABI PRISM™ Optical Adhesive Cover Starter Kit 20 Covers Includes 20 ABI PRISM Optical Adhesive Covers, an Applicator, and a ABI PRISM Optical Cover Compression Pad. 4311971 4323032 ABI PRISM™ Optical Adhesive Covers 100 Covers ABI PRISM™ Optical Caps, 8 Caps/Strip 300 Strips/ Pkg 2400 Caps/ Pkg 384-Well Optical Reaction Plates 4309849 ABI PRISM™ 384-Well Clear Optical Reaction Plate with Barcode (code 128) 50 Plates 4326270 10-Pack, ABI PRISM™ 384-Well Clear Optical Reaction Plate with Barcode (code 128) 500 Plates Includes 10 ABI PRISM 384-Well Clear Optical Reaction Plate with Barcode (P/N 4309849). 96-Well Optical Reaction Plates 4306737 ABI PRISM™ 96-Well Optical Reaction Plate with Barcode (code 128) 20 Plates 4326659 25-Pack, ABI PRISM™ 96-Well Optical Reaction Plate with Barcode (code 128) 500 Plates Includes 25 ABI PRISM 96-Well Optical Reaction Plate with Barcode (P/N 4306737). 4314320 ABI PRISM™ 96-Well Optical Reaction Plate with Barcode (code 128) and ABI PRISM™ Optical Adhesive Covers 100 Plates 100 Covers Includes 100 ABI PRISM Optical Adhesive Covers (P/N 4311971) and 5 ABI PRISM 96-Well Optical Reaction Plate with Barcode packages (P/N 4306737). Miscellaneous 4312063 MicroAmp® Splash Free Support Base for 96-Well Reaction Plates 10 Bases Kits, Reagents and Consumables D-3 Instrument The following sequence detection kits and reagents are used to perform routine Maintenance and maintenance on and verify the function of the ABI PRISM 7900HT Sequence Detection Verification System. For more information about the use of the kits below, see Chapter 7, “System Maintenance.” Part Number Description Quantity Sequence Detection Systems Spectral Calibration Kits 4328639 ABI PRISM™ 7900HT Sequence Detection Systems 96-Well Spectral Calibration Kit 3 x 96-Well Plates Includes three ABI PRISM Optical 384-Well Reaction Plates: one preloaded and sealed Background plate, and two preloaded and sealed Spectral Calibration plates containing eight separate dye standards (FAM™, JOE™, NED™, ROX™, SYBR® Green, TAMRA™, TET™, VIC™). 4323977 Sequence Detection Systems 384-Well Spectral Calibration Kit 2 x 384-Well Plates Includes two ABI PRISM Optical 384-Well Reaction Plates: one preloaded and sealed Background plate, and one preloaded and sealed Spectral Calibration plate containing eight separate dye standards (FAM, JOE, NED, ROX, SYBR Green, TAMRA, TET, VIC). TaqMan RNase P Instrument Verification Plates 4310982 TaqMan® RNase P Instrument Verification Plate Includes one ABI PRISM Optical 96-well Reaction Plate pre-loaded and sealed with Sequence Detection primers and TaqMan® probe to detect and quantitate genomic copies of the human RNase P gene. 4323306 TaqMan® RNase P 384-Well Instrument Verification Plate Includes one ABI PRISM Optical 384-well Reaction Plate preloaded and sealed with complete Sequence Detection primers and TaqMan® probe to detect and quantitate genomic copies of the human RNase P gene. D-4 Kits, Reagents and Consumables 1 x 96-Well Plate 1 x 384-Well Plate TaqMan For the latest information on TaqMan PDARs covering gene expression quantification Pre-Developed and allelic discrimination, visit the TaqMan PDAR list on the Applied Biosystems web Assays and Reagents site at: ♦ www.appliedbiosystems.com/pdarlist Custom To order custom oligonucleotides: Oligonucleotide ♦ Visit the Applied Biosystems Online Store (http://store.appliedbiosystems.com), or Synthesis ♦ Email Applied Biosystems with your order ([email protected]) Part Number Description TaqMan® MGB Probes (5’-Fluorescent label: 6-FAM, VIC or TET) 4316034 5,000-6,000 pmols 4316033 15,000-25,000 pmols 4316032 50,000-100,000 pmols TaqMan® Probes (5’-Fluorescent label: 6-FAM, VIC or TET; 3’-label: TAMRA) 450025 5,000-6,000 pmols 450024 15,000-25,000 pmols 450003 50,000-100,000 pmols Sequence Detection Primers 4304970 Minimum 4,000 pmols purified for sequence detection 4304971 Minimum 40,000 pmols purified for sequence detection 4304972 Minimum 130,000 pmols purified for sequence detection Kits, Reagents and Consumables D-5 References E E Bloch, W. 1991. A biochemical perspective of the polymerase chain reaction. Biochemistry 30:2735–2747. Förster, V. Th., 1948, Zwischenmolekulare Energie-wanderung und Fluoreszenz. Annals of Physics (Leipzig) 2: 55-75. Higuchi, R., Dollinger, G., Walsh, P.S., and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences. BioTechnology 10:413-417. Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. 1993. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. BioTechnology 11:1026-1030. Lakowicz, J.R. 1983. Chapter 10. Energy Transfer. In: Principles of Fluorescent Spectroscopy, Plenum Press, N.Y. pp, 303-339. Lee, L.G., Connell, C.R., and Bloch, W. 1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucl. Acids Res. 21: 3761-3766. Livak, K.J., Flood, S.J.A., Marmaro, J., and Mullah, K.B., inventors; Applied Biosystems (Foster City, CA), assignee. 2 Mar. 1999. Hybridization assay using self-quenching fluorescence probe. United States patent 5,876,930. Livak, K.J., Marmaro, J., and Todd, J.A. 1995. Towards fully automated genome-wide polymorphism screening [letter]. Nat. Genet. 9:341–342. Martens, H. and Naes, T., 1989. In: Multivariate Calibration, John Wiley & Sons, Chichester. References E-1 Contacting Technical Support F F Services and Support Applied Biosystems To access the Applied Biosystems Web site, go to: Web Site http://www.appliedbiosystems.com At the Applied Biosystems Web site, you can: ♦ Search through frequently asked questions (FAQs) ♦ Submit a question directly to Technical Support ♦ Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents ♦ Download PDF documents ♦ Obtain information about customer training ♦ Download software updates and patches In addition, the Applied Biosystems Web site provides a list of telephone and fax numbers that can be used to contact Technical Support. Contacting Technical Support F-1 Limited Warranty Statement G G Warranty Statement PE Corporation (NY), through its Applied Biosystems Group (“Applied Biosystems“) warrants to the customer that, for a period ending on the earlier of one year from the completion of installation or fifteen (15) months from the date of shipment to the customer (the “Warranty Period”), the ABI PRISM ® 7900 HT Sequence Detection System purchased by the customer (the “Instrument”) will be free from defects in material and workmanship, and will perform in accordance with the installation specifications set forth in the system specifications sheet which accompanies the instrument or which is otherwise available from an Applied Biosystems sales representative. During the Warranty Period, if the Instrument's hardware becomes damaged or contaminated or if the Instrument otherwise fails to meet the Specifications, Applied Biosystems will repair or replace the Instrument so that it meets the Specifications, at Applied Biosystems' expense. However, if the thermal cycling module becomes damaged or contaminated, or if the chemical performance of the Instrument otherwise deteriorates due to solvents and/or reagents other than those supplied or expressly recommended by Applied Biosystems, Applied Biosystems will return the Instrument to Specification at the customer's request and at the customer's expense. After this service is performed, coverage of the parts repaired or replaced will be restored thereafter for the remainder of the original Warranty Period. This Warranty does not extend to any Instrument or part which has been (a) the subject of an accident, misuse, or neglect, (b) modified or repaired by a party other than Applied Biosystems, or (c) used in a manner not in accordance with the instructions contained in the Instrument User's Manual. This Warranty does not cover the customer-installable accessories or customer-installable consumable parts for the Instrument that are listed in the Instrument User's Manual. Those items are covered by their own warranties. Applied Biosystems' obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures of the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty Period. All repairs and replacements under this Warranty will be performed by Applied Biosystems on site at the Customer's location at Applied Biosystems's sole expense. Limited Warranty Statement G-1 No agent, employee, or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation, representation, or warranty concerning the Instrument that is not contained in Applied Biosystems's printed product literature or this Warranty Statement. Any such affirmation, representation or warranty made by any agent, employee, or representative of Applied Biosystems will not be binding on Applied Biosystems. Applied Biosystems shall not be liable for any incidental, special, or consequential loss, damage or expense directly or indirectly arising from the purchase or use of the Instrument. Applied Biosystems makes no warranty whatsoever with regard to products or parts furnished by third parties. This Warranty is limited to the original location of installation and is not transferable. THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES, INCLUDING, WITHOUT LIMITATION, ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS. G-2 Limited Warranty Statement Index Symbols > symbol 3-4 Numerics 7900HT instrument, See instrument 9600 emulation mode 4-14 A ABI Prism 7700 Sequence Detection System, emulating 4-14 ABI PRISM SDS Single Plate (*.sds) files See plate documents ABI PRISM SDS Template Document (*.sdt) files See templates absolute quantification about 6-4 to 6-5 analyzing data 6-7 to 6-14 assay development guidelines C-6 procedure checklist 6-7 selecting and preparing standards C-6 setting up (procedure checklist) 4-3 troubleshooting 8-11 adding bar code to a plate document 3-19, 4-19 custom dyes to the pure dye set 7-21 detector tasks to a plate document 4-11 detectors to a plate document 4-8 markers to a plate document 4-10 plate documents to the plate queue 4-31, 4-35 sample names to a plate document 4-19 adjusting analysis options for absolute quantification 6-8 display settings 4-17 method step parameters 4-15 plate-sensor switch 7-28 to 7-31 adjustment knob 7-27 air bubbles 8-5 aligning fixed-position bar code reader 7-40 to 7-42 plate handler 7-32 to 7-39 allele calls about 5-12 calling 5-11 scrutinizing 5-13 DRAFT December 11, 2001 8:32 pm 4317596IX.fm allelic discrimination about 5-4 to 5-6 analyzing run data 5-8 to 5-15 assay development guidelines C-5 maximizing throughput 4-2 procedure checklist 5-8 setting up (procedure checklist) 4-4 thermal cycling on the 7900HT instrument troubleshooting 8-13 Allelic Discrimination Plot about 5-10 calling alleles 5-11 datapoint cluster variations 5-5 exporting as a graphic file B-8 exporting data as a text file B-9 genotypic segregation 5-6 outliers 5-6 scrutinizing allele calls 5-13 amplification curve about A-7 Geometric (Exponential) Phase A-7 Linear Phase A-8 Plateau Phase A-8 Amplification Plot exporting as a graphic file B-8 exporting data as a text file B-9 setting the baseline 6-10 setting the threshold 6-11 visualizing outliers 6-12 analyzing absolute quantification data 6-7 to 6-14 allelic discrimination data 5-8 to 5-15 background data 7-16 dissociation curve data 6-20 to 6-24 pure dye data 7-20 applying detector tasks 4-11 detectors to a plate document 4-8 markers to a plate document 4-10 sample names 4-19 archiving SDS files 7-46 assay development guidelines absolute quantification C-2 to C-4, C-6 allelic discrimination C-2 to C-5 attention words, definitions 1-2 automation accessory components 7-27 fixed-position bar code reader See fixed-position bar code reader plate handler, See plate handler 4-14 Index-1 A(continued) Automation Controller software about 2-14, 4-34 adding plates to the plate queue 4-35 configuring for operation 4-37 ejecting a plate 4-38 launching 4-34 monitoring instrument progress 4-38 removing plates from the plate queue 4-35 starting the plate queue 4-38 stopping the plate queue 4-38 B background run 7-13 to 7-16 about 7-13 constructing a background plate 7-15 creating a plate document 7-14 extracting 7-16 preparing a background plate 7-15 troubleshooting 8-8 when to perform 7-13 bar code information entering into a plate document 3-19, 4-19 bar code readers fixed position, See fixed-position bar code reader hand held, See hand-held bar code reader baseline about A-10 configuring value for automatic analysis 6-8 setting manually 6-10 beam splitter See instrument, optics system Block readout (from the Real Time tab) 4-25 bold text, convention 3-4 C calibrating the 7900HT instrument adjusting the plate-sensor switch 7-28 aligning the fixed-position bar code reader aligning the plate handler 7-32 performing background run 7-13 performing pure dye (spectral) run 7-17 CAUTION attention word, definition 1-2 changing gripper finger pads 7-43 pane, view, and plot sizes 3-15 sample block module 7-4 charged coupled device array See instrument, optics system checklists absolute quantification 6-7 allelic discrimination 5-8 dissociation curve 6-20 plate document setup absolute quantification 4-3 allelic discrimination 4-4 dissociation curve 4-4 chemical safety 1-3 Index-2 7-40 chemistry non-optimized 8-5 SYBR Green A-3 TaqMan A-2 troubleshooting 8-4 to 8-7 cleaning gripper finger pads 7-43 sample block wells 7-12 closing instrument tray from the Automation Controller software 4-38 from the SDS software 4-27 plate documents 3-14 Zymark Twister software 7-39 comments adding to a detector 4-7 adding to a plate document 4-19 computer about 2-7 hard drive partitions 2-7 maintaining 7-46 minimum system requirements 2-7 troubleshooting 8-14 turning ON 3-5 configuring the 7900 instrument for 9600 emulation 4-14 consumables 384-Well Optical Reaction Plates D-3 96-Well Optical Reaction Plates D-3 improper or damaged plastics 8-7 Optical Adhesive Covers D-3 Optical Cap Strips D-3 writing on reaction plates 8-6 contamination decontaminating the sample block 7-11 fluorescent, common sources 8-6 isolating on the sample block module 8-8 contextual menus, using 3-20 copying detectors to a plate document 4-8 markers to a plate document 4-10 Cover readout (from the Real Time tab) 4-25 creating detectors 4-7 markers 4-9 plate documents 3-12, 4-6 for running background plates 7-14 for running custom pure dyes 7-22 for running pure dye plates 7-18 from a template 4-18, 4-32 CT, See threshold cycle custom pure dyes adding to the pure dye set 7-21 to 7-23 creating a custom pure dye plate 7-21 customer support, See technical support cycle set, adding to a method 4-15 D DANGER attention word, definition 1-2 data collection A-5 data type definitions (exportable) B-9 decontaminating the sample block module 7-11 to 7-12 defragmenting the hard drive 7-47 deleting, steps from a method 4-15 detector tasks about 4-11 applying 4-11 importing into a plate document B-2 detectors about 4-7 applying to a plate document 4-8 copying to a plate document 4-8 creating 4-7 importing into a plate document B-2 tasks, See detector tasks determining melting temperatures 6-23 disconnecting the SDS software 4-28 display settings, adjusting 4-17 dissociation curve about 6-18 analyzing 6-20 to 6-24 definitions of the Tm value 6-22 procedure checklist 6-20 programming a temperature ramp 4-16 setting up (procedure checklist) 4-4 Dissociation Plot about 6-22 exporting as a graphic file B-8 exporting data as a text file B-9 E ejecting a plate from the 7900HT instrument 4-27, 4-38 eliminating outliers 6-12 wells from use 6-12 emission filter See instrument, optics system end-point runs about 5-2 allelic discrimination, See allelic discrimination entering bar code information 3-19, 4-19 using the Template Batch utility 4-32 comments into a detector 4-7 into a plate document 4-19 sample names into a plate document 4-19 exportable data type definitions B-9 exporting data from a plate document 3-13, B-9 plots and views as graphic files B-8 F finger pads cleaning 7-43 replacing 7-43 fixed-position bar code reader aligning 7-40 to 7-42 connections 2-11 location 2-8 specification 2-8 fluorescent contamination 8-6 detection system 2-11, A-4 fluorogenic probe about A-2 designing C-2 to C-3 G genotypic segregation of datapoints (Allelic Discrimination Plot) 5-6 Geometric (Exponential) Phase A-7 grating, See instrument, optics system grey dividing line, using 3-15 grid, See plate grid gripper 7-27 guidelines assay development absolute quantification C-6 allelic discrimination C-5 loading the plate handler 4-36 setting the baseline 6-10 setting the threshold 6-11 TaqMan probe design C-2 to C-3 H hand-held bar code reader connections 2-11 location 2-8 specification 2-8 using 3-19 hard drive defragmenting 7-47 partitions 2-7 heated clamp 2-4 help background information 3-3 using the SDS Online Help 3-3 hold, adding to a method 4-15 hotkey combinations, using 3-20 I Important attention word, definition 1-2 importing setup table data 3-14, B-2 imprecise pipetting 8-5 improper threshold setting 8-4 Index-3 I(continued) M installing plate adapter 7-9 sample block module 7-4 SDS software 7-48 the operating system software 7-47 instrument 2-4 about 2-2 to 2-11 external components 2-3 firmware 2-14 internal components 2-4 loading plates 4-24 maintaining 7-2 optics system 2-6, A-4 safety labels 1-2 status lights 3-6 supported runs and chemistries 2-2 troubleshooting 8-14 turning ON 3-5 instrument tray 4-38 opening and closing from the Automation Controller software 4-38 from the SDS software 4-27 replacing the plate adapter 7-9 irreproducibility causes 8-4 to 8-7 J Java Runtime Environment, about 2-14 K keyboard shortcuts, using 3-20 L labels 1-2 laser source, See instrument, optics system launching Automation Controller software 4-34 SDS software 3-7 LAVA software about 2-14 aligning the fixed-position bar code reader 7-40 to 7-42 launching 7-40 LDHost software, See LAVA software learning to use the SDS software 3-11 lights, See instrument, status lights limited warranty statement G-1 Linear Phase of the amplification curve A-8 loading plates into the instrument 4-24 into the plate handler stacks 4-37 low copy templates 8-7 Index-4 maintenance schedule 7-2 manual, See user guide marker inspector 3-22 markers about 4-9 applying to a plate document 4-10 copying to a plate document 4-10 creating 4-9 importing into a plate document B-2 master mixes preparing C-4 using 8-5 maximizing, instrument throughput 4-2 maximizing/minimizing panes, views, and plots melting temperature definition 6-22 determining 6-23 methods about 4-13 to 4-16 adding a hold, cycle set, or step 4-15 temperature ramp 4-16 adjusting step parameters 4-15 configuring data collection options 4-15 programming 4-14 to 4-16 removing a step 4-15 setting the sample volume 4-15 multicomponenting A-5 3-15 N no template control (NTC) detector task applying 4-11 NTC calls applying 5-12 verifying 5-13 normalization of reporter signals A-6 normalized reporter signal A-6 Note attention word, definition 1-2 O opening instrument tray from the Automation Controller software 4-38 from the SDS software 4-27 plate documents 3-13 operating system supported 2-7 upgrading 7-47 operating the 7900HT instrument power switch 3-5 optimizing primer and probe concentrations C-4 outliers absolute quantification 6-12 allelic discrimination 5-6 P panes hiding 3-16 maximizing/minimizing 3-15 resizing 3-15 showing 3-16 passive reference setting 4-12 use in multicomponent analysis A-5 PCR 5´ Nuclease Assay A-2 Geometric (Exponential) Phase A-7 kinetic analysis of A-7 Linear Phase A-8 Plateau Phase A-8 SYBR Green Chemistry A-3 pipetting errors 8-5 pipettors, using 8-5 plate adapter, changing 7-9 plate document information applying to a plate document 4-19 plate documents 4-6 about 3-11 adding to the plate queue 4-31, 4-35 applying detector tasks 4-11 applying sample names 4-19 assigning standard quantities 4-12 closing 3-14 configuring document information 4-19 copying detectors 4-8 copying markers 4-10 creating 3-12, 4-6 from a template 4-18, 4-32 exporting 3-13 importing setup data 3-14, B-2 methods, See methods opening 3-13 programming methods 4-13 removing from the plate queue 4-35 running in batches 4-34 individually 4-20 saving 3-12 as a single plate file 4-22 as a template file 4-17 setting Sample Volume 4-15 setting the passive reference 4-12 plate grid about 3-21 eliminating wells from use 6-12 selecting wells 3-17 viewing well information 3-16 zooming 3-18 plate handler 2-9, 7-27 aligning 7-32 to 7-39 arm adjustment knob 7-27 gripper 7-27 plate-sensor switch 7-27 cleaning the finger pads 7-43 plate stack positions 7-27 replacing the finger pads 7-43 turning ON 3-5 plate queue adding plate documents from the Automation Controller software 4-35 from the SDS software 4-31 using the Template Batch utility 4-32 removing plate documents 4-35 starting 4-38 stopping 4-38 plate stacks loading plates 4-36, 4-37 placing in use 4-37 positions 7-27 Plateau Phase of the amplification curve A-8 plates running batches 4-34 individually 4-23 types, See consumables plate-sensor switch 7-27 adjusting 7-28 to 7-31 plots hiding 3-16 maximizing/minimizing 3-15 resizing 3-15 showing 3-16 Post readout (from the Plate Read tab) 4-25 precision causes of low precision 8-4 to 8-7 preparing master mixes C-4 plate for a run 4-23 primer and probe concentrations optimizing C-4 programming methods for absolute quantification 4-14 for allelic discrimination 4-14 for dissociation curve analysis 4-16 temperature ramp 4-16 pure dye plate constructing for custom dyes 7-21 preparing for use 7-19 pure dye run about 7-17 creating a plate document 7-18 extracting data 7-20 performing 7-19 troubleshooting 8-10 when to perform 7-17 Index-5 Q quantifying probes and primers C-4 standards for absolute quantification C-6 quantitative RT-PCR about 6-2 absolute, See absolute quantification types of 6-2 quantities, applying to a plate document 4-12 R R2 readout (from the Standard Curve Plot) 6-14 reagents custom oligonucleotides D-5 non-Applied Biosystems PCR reagents 8-7 TaqMan Pre-Developed Assays and Reagents D-5 TaqMan RNase P Instrument Verification Plates D-4 re-connecting the SDS software 4-28 removing outliers 6-12 plate documents from the plate queue 4-35 steps from a method 4-15 wells from use 6-12 Rep readout (from the Real Time tab) 4-25 replacing gripper finger pads 7-43 sample block module 7-4 reporter signal normalization A-6 resizing panes, views, and plots 3-15 restacking plates 4-37 Rn, See normalized reporter signal running batches of plates 4-34 single plate 4-23 S safety chemical hazards 1-3 chemical waste 1-3 labels, safety 1-2 laser exposure 1-2 waste disposal 1-4 waste profiles 1-4 sample block locking bar 7-6 sample block locking bolt 7-6 sample block module about 2-5 cleaning sample block module wells 7-12 contamination 8-7 replacing 7-4 sample names adding to a plate document 4-19 importing into a plate document B-2 Sample readout (from the Real Time tab) 4-25 sample volume setting 4-15 saturation, signal 7-21 saving Index-6 plate documents 3-12, 4-22 as template files 4-17 scrutinizing allele calls 5-13 SDS software about 2-14 disconnecting 4-28 installing 7-48 launching 3-7 learning to use the software 3-11 re-connecting 4-28 upgrading 7-48 selecting wells from the plate grid 3-17 setting baseline values 6-10 threshold value 6-11 setup table file about B-4 example files B-4 exporting B-9 importing into a plate document B-2 structure B-5 signal saturation 7-21 Slope readout (from the Standard Curve Plot) 6-14 spectral calibration, See pure dye runs Stage readout (from the Real Time tab) 4-25 Standard Curve Plot about 6-14 exporting as a graphic file B-8 exporting data as a text file B-9 standards quantifying for absolute quantification C-6 selecting for absolute quantification C-6 starting plate queue 4-38 run from the SDS software 4-24 State readout (from the Real Time tab) 4-25 Status readout (from the Real Time tab) 4-25 step adding to a method 4-15 Step readout (from the Real Time tab) 4-25 stopping plate queue 4-38 run from the SDS software 4-26 Support F-1 SYBR Green 1 Dye about A-3 T table pane about 3-21 exporting B-9 TaqMan fluorogenic probe about A-2 designing C-2 to C-3 outliers 6-12 T(continued) TaqMan RNase P Instrument Verification Plates about 7-24 analyzing 7-26 kits D-4 preparing a plate document 7-25 running 7-24, 7-26 tasks, See detector tasks technical support F-1 templates about 3-11 creating a single plate document 4-18 creating multiple plate documents 4-32 saving as 4-17 thermal cycler block, See sample block module thermal cycling protocol, See methods threshold about A-10 configuring for automatic analysis 6-8 improper setting 8-4 setting manually 6-11 threshold cycle calculation A-10 relationship to PCR product A-11 Time readout (from the Real Time tab) 4-25 Time Remaining readout (from the Real Time tab) 4-25 Tm, See melting temperature troubleshooting 8-2 to 8-17 7900HT Instrument 8-14 background runs 8-8 chemistry problems 8-4 to 8-7 computer 8-14 end-point runs 8-13 fixed-position bar code reader 8-17 pure dye runs 8-10 real-time runs 8-11 SDS software 8-14 Zymark Twister Microplate Handler 8-17 turning ON the ABI PRISM 7900HT Sequence Detection System 3-5 W WARNING attention word, definition warranty statement G-1 well inspector 3-22 1-2 Y Y Inter readout (from the Standard Curve Plot) 6-14 Z zooming allelic discrimination plot 5-11 plate grid wells 3-18 Zymark Twister Microplate Handler See plate handler Zymark Twister software about 2-14 aligning the plate handler 7-32 to 7-39 closing 7-39 launching 7-32 testing the plate sensor switch 7-30 U upgrading operating system software SDS software 7-48 user guide conventions 3-4 organization of terminology 3-3 7-47 V viewing, well information 3-16 views hiding 3-16 maximizing/minimizing 3-15 resizing 3-15 showing 3-16 visualizing Index-7 Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free (In North America): +1 800.345.5224 Fax: +1 650.638.5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches into 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our web site at www.appliedbiosystems.com or to the Technical Support and Training appendix in this document. www.appliedbiosystems.com Applera Corporation is committed to providing the world’s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Printed in the USA, 12/2001 Part Number 4317596 Rev. A4 ABI PRISM® 7900HT Sequence Detection System User Guide